Mutant human factor ix with an increased resistance to inhibition by heparin

ABSTRACT

The present invention is related to a novel composition of matter and methods of using the same. More particularly, the invention describes mutant human factor IX which has an increased resistance to inhibition by heparin. Methods of making and using this composition for the therapeutic intervention of hemophilia are disclosed.

[0001] This application claims benefit of U.S. Provisional ApplicationSerial No. 60/248,326 filed Nov. 14, 2000, which is incorporated hereinby reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention provides methods and compositions for usein the treatment of blood coagulation disorders. More particularly, thepresent invention describes mutant human factor IX compositions for usein the therapeutic intervention of hemophilia B.

BACKGROUND OF THE INVENTION

[0003] Hemophilia B (also known as Christmas disease) is one of the mostcommon inherited bleeding disorders in the world. It results indecreased in vivo and in vitro blood clotting activity and requiresextensive medical monitoring throughout the life of the affectedindividual. In the absence of intervention, the afflicted individual maysuffer from spontaneous bleeding in the joints, which produces severepain and debilitating immobility; bleeding into muscles results in theaccumulation of blood in those tissues; spontaneous bleeding in thethroat and neck may cause asphyxiation if not immediately treated;bleeding into the urine; and severe bleeding following surgery, minoraccidental injuries, or dental extractions also are prevalent.

[0004] To the extent that the present invention relates to interventionof blood clotting disorders, a brief discussion of the biologicalfactors and/or mechanisms involved in blood clotting is warranted. Ablood clot is essentially a gelatinous mass, which seals blood vesselsthat have sustained an injury. Conversion of fluid blood to a blood clotinvolves the conversion of soluble fibrinogen, which is present inplasma, to the insoluble gelatinous blood clot, composed primarily ofcross-linked fibrin. The conversion of fibrinogen to fibrin is theprimary end result of a multi-step process referred to as the bloodcoagulation cascade. This-cascade is a highly regulated process thatinvolves the sequential proteolytic conversion of serine proteases fromzymogen to active conformations, and subsequent formation ofcalcium-dependent phospholipid-bound enzyme complexes with specificprotein cofactors. Normal in vivo blood coagulation at minimum requiresthe serine proteases factors II (prothrombin), VII, IX, X and XI(soluble plasma proteins); cofactors including the transmembrane proteintissue factor and the plasma proteins factors V and VIII; fibrinogen,the transglutaminase factor XIII, phospholipid (including activatedplatelets), and calcium. Additional proteins including kallikrein, highmolecular weight kininogen, and factor XII are required for some invitro clotting tests, and may play a role in vivo under pathologicconditions. The coagulation cascade is regulated by thethrombomodulin-protein C pathway, the fibrinolysis pathway, tissuefactor pathway inhibitor, and the serpin antithrombin III. Importantly,the inhibition of several coagulation proteases by antithrombin III(including factor IXa) is markedly accelerated by the anticoagulant drugheparin, as well as structurally similar heparan sulfate on theendothelial surface.

[0005] Upon injury, thrombocytes, in the presence of von WillebrandFactor (a component of clotting Factor VIII), cling to the collagen ofinjured connective tissue by adhesion. The thrombocytes change theirform and develop protrusions, and in addition to this, their outermembrane facilitates the adhesion of further thrombocytes. Thereafter,various substances are released from granula of these cells, whichresults in vessel constriction as well as accumulation and activation ofother factors of plasma blood clotting.

[0006] In hemophilia, blood clotting is disturbed by a lack of certainplasma blood clotting factors. Hemophilia B is caused by a deficiency infactor IX that may result from either the decreased synthesis of thefactor IX protein or a defective molecule with reduced activity. Thetreatment of hemophilia occurs by replacement of the missing clottingfactor by exogenous factor concentrates highly enriched in factor IX.However, generating such a concentrate is fraught with technicaldifficulties as described below.

[0007] Factor IX, like other clotting factors, is naturally produced asa precursor molecule having an additional pre-pro-sequence at theN-terminus. The pre-pro-sequence represents a signal sequence thatcauses the oriented transport of this protein in the cell. When thepre-pro Factor DC protein is secreted from the cell the pre-sequence iscleaved. The pro-sequence consists of about 15 to 18 amino acids andserves as a recognition sequence in carboxylation of glutamic acidresidues to 4-carboxy-L-glutamic acid. After successful carboxylation,the pro-sequence is also cleaved. If the pro-sequence is not cleaved oronly incompletely cleaved, only low activity clotting factors result.Human factor IX has a molecular weight of about 55,000 Dalton; when itspro-sequence is present the molecular weight is increased by about 2000Dalton.

[0008] Purification of factor IX from plasma almost exclusively yieldsactive factor IX. However, such purification of factor IX from plasma isvery difficult because factor IX is only present in low concentration inplasma [5 μg/mL; Andersson, Thrombosis Research 7: 451-459 (1975)].Efforts to produce recombinant factor IX have led to products with onlylow levels of activity [Kaufman et al., J. Biol Chem 261: 9622-9628(1986); Busby et al., Nature 316: 217-273 (1985); Rees et al., EMBO J 7:2053-2061 (1988)]. This can be traced back to an incomplete cleavage ofthe pro-sequence [Meulien et al., Prot Engineer 3: 629-633 (1990)]because a mixture of recombinantly produced pro-factor IX and factor IXis present in cell supernatants.

[0009] The in vivo activity of exogenously generated factor IX islimited both by protein half-life and inhibitors of coagulation,including antithrombin III. An additional factor that limits the use ofexogenously generated factor IX in an effective therapeutic protocol isthat endogenous heparan sulfate/heparin greatly inhibits the activity offactor IX that is used in the existing therapies for hemophilia B.

[0010] Heparin can inhibit factor IXa activity in the intrinsic tenasecomplex (factor IXa-factor VIIIa) directly, or markedly accelerateinhibition of factor IXa by antithrombin III. Heparan sulfate, achemically similar glycosaminoglycan to heparin, is expressed widely inthe body including the endothelial surface, where it has beendemonstrated to accelerate inhibition of coagulation proteases byantithrombin III. Similarly, it may directly inhibit intrinsic tenaseactivity at sites of injury, limiting the in vivo activity of factorIXa. Thus, a mutant factor IXa that is resistant to the effects ofheparin/heparan sulfate and retains in vitro clotting activity may haveenhanced in vivo activity. Similar protein engineering approaches havebeen used to enhance the therapeutic efficacy of other serine proteases,including improvement of the fibrin binding properties of tissueplasminogen activator by mutagenesis.

[0011] Thus, there is a need for mutant factor IX, which has a reducedaffinity for heparin but retains it anti-clotting activity, and remainsactive when administered as part of a therapeutic regimen for hemophiliaB.

SUMMARY OF THE INVENTION

[0012] The present invention provides novel mutant forms of factor IXthat may be used in the therapeutic intervention of hemophilia B. In apreferred embodiment, the present invention provides a mutant humanfactor IX comprising a mutation in the heparin binding domain of factorIX, which decreases its affinity for heparin, as compared to wild-typehuman factor IX. By heparin binding domain, the present invention refersto that site on the factor IX protein that binds to and interacts withheparin. In more specific embodiments, the mutation is a mutation of theamino acid residue 233, 230, 239, 241, 87, 91, 98, 101, or 92 ofwild-type human factor IX. In preferred embodiments, it is contemplatedthat one or more of these residues is mutated. In additionalembodiments, it is contemplated that the mutation further comprises asubstitution of arginine 170 of the wild-type human factor IX for analanine. The numbering system employed herein is the chymotrypsinnumbering system well known to those of skill in the art [Bajaj andBirktoft, Meth Enzymol 222:96-128 (1993)] and is also depicted herein inFIG. 3.

[0013] A preferred mutant human factor IX of the present invention has amutation of the amino acid located at residue number 233 of wild-typehuman factor IX, wherein said mutation decreases the affinity of saidmutant human factor IX for heparin as compared to wild-type human factorIX. More particularly, the mutation is a substitution of the arginine atposition 233 to any other amino acid. In still more specificembodiments, it is contemplated that the arginine at position 233 issubstituted with an alanine. Alternatively, the arginine at 233 or anyother arginine in the heparin binding domain is replaced with aglutamate.

[0014] Another aspect of the present invention describes a method oftreating a subject having hemophilia comprising administering to saidsubject a composition comprising a mutant human factor IX of the presentinvention, in an amount effective to promote blood clotting in saidsubject. Further methods contemplate administering to the subject acomposition comprising one or more additional blood clotting factorsother than said mutant human factor IX. In particular embodiments, thesubject suffering from hemophilia is suffering from hemophilia B.

[0015] Additional methods of treating hemophilia in a mammal also arecontemplated. Such methods comprise providing an expression constructcomprising a polynucleotide encoding a mutant factor IX of the presentinvention, operably linked to a promoter; and administering in a mammalan amount of the expression construct effective to allow the mutantfactor IX to be expressed at levels having a therapeutic effect on themammal. The indicia of a therapeutic effect of a given hemophiliatherapy are well known to those of skill in the art. For example, in thepresent invention, the therapeutic effect is an increased resistance offactor IX to inhibition by heparin. More particularly, the therapeuticeffect is a decrease in the blood clotting time of said mammal ascompared to the blood clotting time of said mammal in the absence ofsaid expression construct. In preferred aspects the expression constructcomprises a viral vector selected from the group consisting of anadenovirus, an adeno-associated virus, a retrovirus, a herpes virus, alentivirus and a cytomegalovirus. In preferred embodiments, theexpression control element is selected from the group consisting of acytomegalovirus immediate early promoter/enhancer, a skeletal muscleactin promoter and a muscle creatine kinase promoter/enhancer.

[0016] In specific embodiments, it is contemplated that the therapeuticcompositions (protein or expression vector compositions) of the presentinvention are administered to at least two sites in the mammal.

[0017] Yet another aspect of the present invention contemplates arecombinant host cell stably transformed or transfected with apolynucleotide encoding a mutant human factor IX of the presentinvention in a manner allowing the expression in the host cell of saidmutant human factor IX. In specific embodiments, such a recombinant hostcell may be employed in methods of for the large scale production of themutant human factor IX protein.

[0018] Also contemplated by the present invention are pharmaceuticalcompositions comprising a mutant human factor IX protein of the presentinvention and a pharmaceutically acceptable carrier, excipient ordiluent. Also contemplated are pharmaceutical compositions comprising anexpression construct comprising a vector having an isolatedpolynucleotide encoding a mutant human factor IX of the instantinvention and a promoter operably linked to said polynucleotide; and apharmaceutically acceptable carrier, excipient or diluent. It iscontemplated that in addition to the mutant human factor IX, thepharmaceutical compositions may comprise additional therapeuticcomponents such as for example, other blood clotting factors andhemophilia therapeutic compositions.

[0019] Other aspects, features and advantages of the present inventionwill be apparent from the detailed description. It should be understoodthat the detailed description presented below, while providing preferredembodiments of the invention, is intended to be illustrative only sincechanges and modification within the scope of the invention will bepossible whilst still providing an embodiment that is within the spiritof the invention as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following drawings form part of the present specification andare included to further demonstrate aspects of the present invention.The invention may be better understood by reference to one or more ofthe drawings in combination with the detailed description of thespecific embodiments presented herein.

[0021]FIG. 1. Purification of recombinant human factor IXa from 293cells. Sliver stained nonreducing 10% SDS-PAGE representing samples frompurification steps. Lane 1 and 10- molecular weight markers, lane 2-conditioned media, lane 3 barium depleted media, lane 4- barium eluate,lane 5-9 fractions 17, 18, 19, 20, and 23 eluted from Mono Q HR 5/5 withcalcium chloride gradient (0-30 mM). The human factor IX isolated usingthis method demonstrated high purity by 10% SDS-PAGE with silverstaining, high specific clotting activity (187 U/mg), and an overallyield of approximately 30% by ELISA.

[0022]FIG. 2. Effect of unfractionated heparin on intrinsic tenaseactivity using recombinant factor IXa in conditioned media. Serum-freemedia incubated for 48 hr following transient transfection of 293 cellswas concentrated by Centricon-30 and activated with 2 nM human factorXIa for 2 hr at 37° C. The intrinsic tenase assay was performed in whichfactor Via was in excess (5 nM), and conditioned media served as theenzyme source. The rate of factor Xa generation in the presence ofincreasing amounts of unfractionated heparin is plotted for wild-type() and R233A (◯) factor IXa. Mock-transfected media demonstrated nosignificant activity. There was an increase in the residual activity inthe plateau phase for the mutant R233A (˜65%) relative to wild-typefactor IXa (˜15%).

[0023]FIG. 3A through FIG. 3C. Contiguous DNA (SEQ ID NO: 1) and aminoacid (SEQ ID NO: 2) sequences of factor IX, including bold numbersunderneath, designating the amino acid sequence corresponding to thechymotrypsin numbering system.

[0024]FIG. 4A and FIG. 4B. The rate of factor Xa generation by 5 nMwild-type () or R233A (◯) factor IXa in the factor IXa-phospholipid (A)and the intrinsic tenase (B) complexes in the presence of increasingamounts of unfractionated heparin. Tie mutant factor Ma R233Ademonstrates increased resistance to inhibition by heparin relative towild-type factor IXa.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Hemophilia B is one of the most prevalent blood clottingdisorders and results from a deficiency of or defect in endogenousfactor IX gene expression or activity, respectively. Therapeuticintervention requires replacement therapy in which the patient isprovided with exogenous factor IX. However, treatment is limited by thecommercial availability of clotting factor and the expense of treatment.Further, factor IX that is isolated from natural sources or that isproduced recombinantly using native sequences is inhibited by endogenousheparin/heparan sulfate in both an antithrombin III-dependent andindependent manner, limiting in vivo activity and half-life of activatedfactor IXa. Hence the presently available replacement therapies areineffective at providing an adequate remedy for the disease.

[0026] The present invention describes a mutant human factor IX that hasan increased resistance to heparin inhibition in vitro as compared towild-type human factor IX. More particularly, the invention describes amutant factor IX that has an Arg to Ala substitution at residue 233(according to the chymotrypsin numbering system, see FIG. 3, and SEQ IDNO: 2). The increased resistance of this mutant human factor IX toheparin means that the mutant human factor IX is a more effectivereplacement therapy for patients suffering from hemophilia B thanadministering wild-type factor IX. Further, it is expected that thismutant human factor IX also may possess an increased in vivo bloodclotting activity as compared to wild-type human factor IX. Methods andcompositions for exploiting the therapeutic potential of these findingsare discussed in further detail herein below.

[0027] A. Role of Factor IX in the Blood Coagulation Cascade.

[0028] Factor IX is a key serine protease that participates in themiddle phase of the blood clotting cascade. Factor IX is activated byeither factor XIa or by factor VIIa-tissue factor in a Ca²⁺ dependentmanner. The activated factor IXa with its cofactor VIIIa, in thepresence of Ca²⁺ and phospholipid, forms the intrinsic tenase complexand is responsible for generating activated Factor X. Factor IX isfunctionally deficient or absent in individuals with the inheriteddisorder hemophilia B.

[0029] Ex vivo modeling of blood coagulation demonstrates that formationof the membrane bound intrinsic tenase (factor IXa-factor VIIIa) andprothrombinase (factor Xa-factor Va) complexes results in a localized,explosive increase in thrombin generation [Lawson et al., J Biol Chem.,269(37):23357-66 (1994); Rand et al., Blood, 88(9):3432-45 (1996)]. Inminimally altered whole blood, the rate limiting factor for thrombingeneration is the activation of factor Xa by the intrinsic tenasecomplex [Rand et al., Blood, 88(9): 3432-45 (1996)]. In a cell-basedsystem containing platelets and monocytes expressing tissue factor,addition of picomolar factor IXa generates significantly more thrombinthan similar concentrations of factor Xa [Hoffman et al., Blood, 86(5):179-801 (1995)]. Omitting either factor IX or factor VIII markedlyreduces the generation of thrombin [Lawson et al., J Biol Clem, 269(37):23357-66(1994)]. Thus, formation and activity of the intrinsic tenasecomplex is critical to the final rate of thrombin generation during thepropagation phase of coagulation. The activity of the intrinsic tenasecomplex appears to be primarily regulated by instability (loss of the A2domain) and proteolytic inactivation of factor VIIIa (by factor IXa)[Fay et al., J Biol Chem, 271(11): 6027-32 (1996)]. The pivotal role ofintrinsic tenase suggests that regulation of this enzyme complex iscritical to maintaining hemostatic balance.

[0030] Heparin has anticoagulant effects prolongation of in vitrocoagulation assays) in plasma which are antithrombin (ATIII) dependent,largely attributed to the acceleration of factor Xa and thrombininhibition [Hirsh et al., Chest 108(4 Suppl) (1995)]. In both thepurified state and in plasma, ATIII inhibits factor IXa at asignificantly slower baseline rate than thrombin and factor Xa [Jordanet al., J Biol Chem, 255(21):10081-90 (1980); Pieters et al., J BiolChem, 263(30):15313-8 (1988); Pieters et al., Blood, 76(3): 549-54(1990)]. However, addition of unfractionated heparin tocontact-activated plasma results in a significant increase in the amountof factor IXa-antithrombin complex formed [McNeely and Griffith, Blood,65(5): 1226-31 (1985)]. Similar to thrombin, heparin acceleration offactor IXa inhibition by antithrombin demonstrates a biphasic doseresponse, and requires high molecular weight oligosaccharides foroptimal rate enhancement [Holmer et al., Biochem J, 193(2): 395-400(1981); Mauray et al., Biochim Biophys Acta, 1387(1-2): 184-94 (1998)].These results suggest a template mechanism for heparin catalysis, inwhich inhibition is accelerated by the binding of protease and inhibitorto the same oligosaccharide chain.

[0031] In addition to accelerating protease inhibition by ATIII, heparinalso inhibits the intrinsic tenase complex in an a ATIII independentmanner [Barrow et al., J Biol Chem, 269(43): 26796-800 (1994)]. Thisinhibition exhibits a partial, noncompetitive pattern, which is notexplained by effects on cofactor stability or assembly of the factorIXa-factor VIIIa complex [Barrow et al., J Biol Chem, 269(43): 26796-800(1994)]. By eliminating potential effects on assembly or stability ofthe complex that would reduce the effective enzyme concentration, theseresults suggest that heparin directly modulates the catalytic activityof the enzyme complex [Barrow et al., J Biol Chem, 269(43): 26796-800(1994)]. From mechanistic studies the inventor has inferred a model inwhich heparin binds to a regulatory site on factor IX. The inventor hasshown that site directed mutagenesis of the heparin binding domain (atposition R233) generated a mutant human factor IX, which when comparedto wild-type factor IX, demonstrated markedly reduced inhibition byheparin. This mutant human factor IX as well as other mutant humanfactor IX proteins in which the regulatory site that binds heparin hasbeen disrupted will be useful in replacement therapy for individualssuffering from hemophilia.

[0032] The amino acid structure of human factor DC is well known tothose of skill in the art, see Bajaj and Birktoft [Meth Enzymol, 222:96-128 (1993)]. Given that the instant invention has shown that it ispossible to generate such a mutant, those of skill in the art will beable to produce other mutants having a similar activity. Similarly, thenucleic acid sequence of the gene encoding factor IX also is well knownto those of skill in the art (see FIG. 3 and SEQ ID NO: 1).

[0033] Of additional interest, recent studies have shown that theendocytic receptor low density lipoprotein receptor-related protein(LRP) was demonstrated to bind factor IXa upon activation from a zymogenform in a two-site binding model with equilibrium dissociation constantsof 27 nM and 69 nM [eels et al., Blood 96(10): 3459-3465 (2000)].Modification of the factor IXa active site, however, did not affectbinding to LRP, suggesting that binding of factor IXa to LRP involves anenzyme exosite. LRP-deficient cells degrade 35% less factor IXa thanLRP-expressing cells, suggesting a role for LRP in the transport offactor IXa to the intracellular degradation pathway. Degradation offactor IXa by proteoglycan-deficient cells proceeded at a rate lowerthan 83% than that of wild-type cells, also suggesting a role forproteoglycans in the binding to LRP. Furthermore, the binding of factorIXa to LRP can be fully inhibited in the presence of either 100 U/mLunfractionated or low molecular weight heparin. In contrast, little, ifany, inhibition was observed in the presence of 100-μg/mL chondroitinsulfate. These data indicate that the heparin-binding domain of factorIXa may contribute to the interaction with LRP. Thus, factor IXaproteins with reduced affinity for heparin may have reduced clearance byLRP-dependent mechanisms, further enhancing their in vivo activity.

[0034] B. Mutant Factor IX

[0035] The present invention contemplates the production of mutant humancoagulation factor IX that has an increased resistance to inhibition byheparin/heparan sulfate by both antithrombin-dependent and independentmechanisms. By mutant human factor IX, the present invention means humanfactor IX in which the wild-type sequence has been mutated.

[0036] Specifically contemplated by the present invention issite-specific mutagenesis of wild-type human factor IX. While the aminoacid sequence variants of the polypeptide can be substitutional mutantsin which the amino acid at a given site is substituted for another,insertional or deletion variants also are contemplated.

[0037] Substitutional variants typically exchange one amino acid of thewild-type for another at one or more sites within the protein, and maybe designed to modulate one or more properties of the polypeptide, suchas stability against proteolytic cleavage, without the loss of otherfunctions or properties. Substitutions of amino acids to maintainactivity or properties preferably are conservative, that is, one aminoacid is replaced with one of similar shape and charge.

[0038] Conservative substitutions are well known in the art and include,for example, the changes of: alanine to serine; arginine to lysine;asparagine to glutamine or histidine; aspartate to glutamate; cysteineto serine; glutamine to asparagine; glutamate to aspartate; glycine toproline; histidine to asparagine or glutamine; isoleucine to leucine orvaline; leucine to valine or isoleucine; lysine to arginine; methionineto leucine or isoleucine; phenylalanine to tyrosine, leucine ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

[0039] Variant polypeptides include those wherein conservativesubstitutions have been introduced by modification of polynucleotidesencoding polypeptides of the invention. Amino acids can be classifiedaccording to physical properties and contribution to secondary andtertiary protein structure. A conservative substitution is recognized inthe art as a substitution of one amino acid for another amino acid thathas similar properties. Exemplary conservative substitutions are set outin the Table A (from WO 97/09433, page 10, published Mar. 13, 1997(PCT/GB96/02197, filed 9/6/96), immediately below. TABLE A ConservativeSubstitutions I SIDE CHAIN CHARACTERISTIC AMINO ACID Aliphatic Non-polarG A P I L V Polar - uncharged C S T M N Q Polar - charged D E K RAromatic H F W Y Other N Q D E

[0040] Alternatively, conservative amino acids can be grouped asdescribed in Lehninger. [Biochemistry, Second Edition; Worth Publishers,Inc. NY: (1975), pp.71-77] as set out in Table B, immediately below.TABLE B Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINOACID Non-polar (hydrophobic) A. Aliphatic: A L I V P B. Aromatic: F W C.Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: S T YB. Amides: N Q C. Sulfhydryl: C D. Borderline: G Positively Charged(Basic): K R H Negatively Charged (Acidic): D E

[0041] As still an another alternative, exemplary conservativesubstitutions are set out in Table C, immediately below. TABLE CConservative Substitutions III Original Residue Exemplary SubstitutionAla (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, ArgAsp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys,Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys(K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro(P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, SerVal (V) Ile, Leu, Met, Phe, Ala

[0042] Also contemplated are non-conservative substitutions, in which anamino acid is replaced with one of different properties. Replacement ofarginine or lysine to glutamate (charge reversal) to disrupt theelectrostatic binding of the protease to heparin, similar to thestrategy used for thrombin-heparin binding, is an example of suchnon-conservative substitutions [Sheehan and Sadler, Proc Nat'l Acad SciUSA, 91(12): 5518-22 (1994)]. Such nonconservative mutations may beuseful in generating further mutants that have increased resistance toheparin inhibition.

[0043] The binding of polyanionic heparin chains to factor IX(a) likelyinvolves interactions with basic amino acid residues on the proteasesurface. The binding of heparin to thrombin, a homologous coagulationprotease, is a highly electrostatic interaction that involves a numberof basic residues in exosite II [Sheehan and Sadler, Proc Nat'l Acad SciUSA, 91(12): 5518-22 (1994); Olson et al., J Biol Chem; 266(10): 6342-52(1991)]. The inventor prepared a three-dimensional structure of humanfactor Ixa by homology using SWISS-MODEL. Based on homology to thethrombin-heparin interaction, basic surface residues (lysine, arginine,or histidine) in the carboxyl-terminus -helix, and the insertion loop80-90 (chymotrypsin numbering) are appropriate targets for mutagenesis.Candidate residues include R87, H91, H92, K98, H101 in the 80-90 loopregion, and K230, R233, K239, and K241 in the carboxyl-terminus helix.It is expected that these mutations will be within the heparin bindingdomain of factor IX. Preferred mutants include single amino acidsubstitutions of alanine for R87, H92, R233, H101, and K241. Otherresidues are those that are within about 5 Å that interact with theseaforementioned residues. The mutations may be combined with asubstitution of R170 to A170 [Chang et al., J Biol Chem, 273(20):12089-94 (1998)]. Of course these residues could also be mutated to anyother residue if desired, so long as the mutation provided a mutanthuman factor IX that was resistant to inhibition by heparin. In otherpreferred aspects, such a mutant human factor IX also retains bloodcoagulation activity. Using such mutagenesis also will allow mapping ofthe heparin binding site, similar to the mapping studies performed forthrombin [Sheehan and Sadler, Proc Nat'l Acad Sci USA, 91(12): 5518-22(1994)]. Further it is contemplated that mutations may be combined toprovide a more dramatic effect on heparin binding and function.

[0044] A preferred embodiment of the present invention contemplatesgenerating a mutation of the arginine at 233. This arginine may bemutated to any amino acid. It should be noted that the mutants of thefactor Ix peptide should have an increased resistance to heparininhibition. Such mutants also may preferably possess an increasedclotting activity.

[0045] In order to construct mutants such as those described above, oneof skill in the art may employ well known standard technologies.Proteins expressed from such mutant can be assayed for appropriateheparin inhibition and/or effect on blood clotting as described infurther detail below.

[0046] A random insertional mutation may also be performed by cuttingthe DNA sequence with a DNase I, for example, and inserting a stretch ofnucleotides that encode, 3, 6, 9, 12 etc., amino acids and religatingthe end. Once such a mutation is made the mutants can be screened forvarious activities presented by the wild-type protein.

[0047] Point mutagenesis also may be employed to identify withparticularity the amino acid residues that are important in particularactivities associated with the heparin binding of factor IX. Thus, oneof skill in the art will be able to generate single base changes in theDNA strand to result in an altered codon and a missense mutation.

[0048] Site-specific mutagenesis is a technique useful in thepreparation of individual peptides, or biologically functionalequivalent proteins or peptides, through specific mutagenesis of theunderlying DNA. The technique further provides a ready ability toprepare and test sequence variants, incorporating one or more of theforegoing considerations, by introducing one or more nucleotide sequencechanges into the DNA. Site-specific mutagenesis allows the production ofmutants through the use of specific oligonucleotide sequences thatencode the DNA sequence of the desired mutation, as well as a sufficientnumber of adjacent nucleotides, to provide a primer sequence ofsufficient size and sequence complexity to form a stable duplex on bothsides of the nucleotide(s) being mutated. Typically, a primer of about17 to 25 nucleotides in length is preferred, with about 5 to about 10matching bases on both sides of the nucleotide(s) being altered.

[0049] The technique typically employs a bacteriophage vector thatexists in both a single-stranded and double-stranded form. Typicalvectors useful in site-directed mutagenesis include vectors such as theM13 phage. These phage vectors are commercially available and their useis generally well known to those skilled in the art. Double-strandedplasmids also are routinely employed in site-directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

[0050] In general, site-directed mutagenesis is performed by firstobtaining a single-stranded vector, or melting of two strands of adouble-stranded vector, which includes within its sequence a DNAsequence encoding the desired protein. An oligonucleotide primer bearingthe desired mutated sequence is synthetically prepared. This primer isthen annealed with the single-stranded DNA preparation, taking intoaccount the degree of mismatch when selecting hybridization conditions,and subjected to DNA polymerizing enzymes such as E. coli polymerase IKlenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation. This heteroduplex vector is then used totransform appropriate cells, such as E. coli cells, and clones areselected that include recombinant vectors bearing the mutated sequencearrangement.

[0051] A PCR-based method for site-directed mutagenesis is particularlypreferred. Overlapping forward (positive strand) and reverse (negativestrand) primers containing the desired mutation and 10-15 matchingnucleotides flanking both sides, are annealed with the denaturedwild-type cDNA in a suitable plasmid vector (i.e. Bluescript®). Thistemplate is then subject to amplification by PCR with a high fidelitythermostable DNA polymerase, the product digested with the restrictionendonuclease Dpn I (to degrade the methylated parental or wild-typeplasmid), and resulting DNA employed for transformation of bacteria.Antibiotic resistant bacterial colonies (containing the plasmid) arethen selected for overnight growth, isolation of plasmid DNA (miniprep),and sequencing to confirm the presence of the mutation.

[0052] C. Recombinant Protein Production.

[0053] Given the above disclosure of mutant human factor IX peptides itwill be possible for one of skill in the art to produce human factor IXpeptides by automated peptide synthesis, by recombinant techniques orboth.

[0054] The mutant factor IX protein of the invention can be synthesizedin solution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. See, for example,Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., PierceChemical Co., (1984); Tam et al., J Amer Chem Soc, 105: 6442 (1983);Merrifield, Science, 232: 341-347 (1986); and Barany and Merrifield, ThePeptides, Gross and Meienhofer, eds, Academic Press, New York, 1-284(1979), each incorporated herein by reference. The active protein can bereadily synthesized and then screened in screening assays designed toidentify reactive peptides.

[0055] Alternatively, a variety of expression vector/host systems may beutilized to contain and express a mutant factor IX coding sequence.These include but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transfected with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with bacterial expression vectors (e.g., Ti orpBR322 plasmid); or animal cell systems. Mammalian cells that are usefulin recombinant protein productions include but are not limited to VEROcells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells(such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and293 cells. Exemplary protocols for the recombinant expression of theprotein are described herein below.

[0056] A yeast system may be employed to generate the mutant peptides orproteins of the present invention. The coding region of the mutantfactor IX cDNA is amplified by PCR. A DNA encoding the yeastpre-pro-alpha leader sequence is amplified from yeast genomic DNA in aPCR reaction using one primer containing nucleotides 1-20 of the alphamating factor gene and another primer complementary to nucleotides255-235 of this gene [Kurjan and Herskowitz, Cell, 30: 933-943 (1982)].The pre-pro-alpha leader coding sequence and mutant factor IX codingsequence fragments are ligated into a plasmid containing the yeastalcohol dehydrogenase (ADH2) promoter, such that the promoter directsexpression of a fusion protein consisting of the pre-pro-alpha factorfused to the mature mutant factor IX polypeptide. As taught by Rose andBroach [Meth Enzymol, 185: 234279, D. Goeddel, ed., Academic Press,Inc., San Diego, Calif. (1990)], the vector further includes an ADH2transcription terminator downstream of the cloning site, the yeast“2-micron” replication origin, the yeast leu-2d gene, the yeast REP1 andREP2 genes, the E. coli beta-lactamase gene, and an E. coli origin ofreplication. The beta-lactamase and leu-2d genes provide for selectionin bacteria and yeast, respectively. The leu-2d gene also facilitatesincreased copy number of the plasmid in yeast to induce higher levels ofexpression. The REP1 and REP2 genes encode proteins involved inregulation of the plasmid copy number.

[0057] The DNA construct described in the preceding paragraph istransformed into yeast cells, using a known method, e.g., lithiumacetate treatment [Stearns et al., Meth Enzymol, 185: 280-297 (1990)].The ADH2 promoter is induced upon exhaustion of glucose in the growthmedia [Price et al., Gene, 55: 287 (1987)]. The pre-pro-alpha sequenceeffects secretion of the fusion protein from the cells. Concomitantly,the yeast KEX2 protein cleaves the pre-pro sequence from the maturemutant factor IX [Bitter et al., Proc Nat'l Acad Sci USA, 81: 5330-5334(1984)].

[0058] Alternatively, mutant factor IX may be recombinantly expressed inyeast using a commercially available expression system, e.g., the PichiaExpression System (Invitrogen, San Diego, Calif.), following themanufacturer's instructions. This system also relies on thepre-pro-alpha sequence to direct secretion, but transcription of theinsert is driven by the alcohol oxidase (AOX1) promoter upon inductionby methanol.

[0059] The secreted mutant human factor IX is purified from the yeastgrowth medium by, e.g., the methods used to purify mutant factor IX frombacterial and mammalian cell supernatants.

[0060] Alternatively, the cDNA encoding mutant factor IX may be clonedinto the baculovirus expression vector pVL1393 (PharMingen, San Diego.Calif.). This mutant factor IX-containing vector is then used accordingto the manufacturer's directions (PharMingen) to infect Spodopterafrugiperda cells in sF9 protein-free media and to produce recombinantprotein. The protein is purified and concentrated from the media using aheparin-Sepharose column (Pharmacia, Piscataway, N.J.) and sequentialmolecular sizing columns (Amicon, Beverly, Mass.), and resuspended inPBS. SDS-PAGE analysis shows a single band and confirms the size of theprotein, and Edman sequencing on a Porton 2090 Peptide Sequencerconfirms its N-terminal sequence.

[0061] Alternatively, the mutant factor IX may be expressed in an insectsystem. Insect systems for protein expression are well known to those ofskill in the art. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The mutantfactor IX coding sequence is cloned into a nonessential region of thevirus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of mutant factor IX willrender the polyhedrin gene inactive and produce recombinant viruslacking coat protein coat. The recombinant viruses are then used toinfect S. frugiperda cells or Trichoplusia larvae in which mutant factorIX is expressed [Smith et al., J Virol, 46: 584 (1983); Engelhard etal., Proc Nat'l Acad Sci USA, 91: 3224-7 (1994)].

[0062] In another example, the DNA sequence encoding the mature form ofthe protein is amplified by PCR and cloned into an appropriate vectorfor example, pGEX-3X (Pharmacia, Piscataway, N.J.). The pGEX vector isdesigned to produce a fusion protein comprisingglutathione-S-transferase (GST), encoded by the vector, and a proteinencoded by a DNA fragment inserted into the vector's cloning site. Theprimers for the PCR may be generated to include, for example, anappropriate cleavage site.

[0063] The recombinant fusion protein may then be cleaved form the GSTportion of the fusion protein. The pGEX-3X/mutant human factor IXconstruct is transformed into E. coli XL-1 Blue cells (Stratagene, LaJolla Calif.), and individual transformants were isolated and grown.Plasmid DNA from individual transformants is purified and partiallysequenced using an automated sequencer to confirm the presence of thedesired mutant human factor IX encoding gene insert in the properorientation.

[0064] While certain embodiments of the present invention contemplateproducing the mutant human factor IX protein using synthetic peptidesynthesizers and subsequent FPLC analysis and appropriate refolding ofthe cysteine double bonds, it is contemplated that recombinant proteinproduction also may be used to produce the mutant human factor IXpeptide compositions. For example, induction of the GST/mutant humanfactor IX fusion protein is achieved by growing the transformed XL-1Blue culture at 37° C. in LB medium (supplemented with carbenicillin) toan optical density at wavelength 600 nm of 0.4, followed by furtherincubation for 4 hours in the presence of 0.5 mM Isopropylβ-D-Thiogalactopyranoside (Sigma Chemical Co., St. Louis Mo.).

[0065] The fusion protein, expected to be produced as an insolubleinclusion body in the bacteria, may be purified as follows. Cells areharvested by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1mM EDTA; and treated with 0.1 mg/mL lysozyme (Sigma Chemical Co.) for 15minutes at room temperature. The lysate is cleared by sonication, andcell debris is pelleted by centrifugation for 10 minutes at 12,000 ×g.The fusion protein-containing pellet is resuspended in 50 mM Tris, pH 8,and 10 mM EDTA, layered over 50% glycerol, and centrifuged for 30 min.at 6000 ×g. The pellet is resuspended in standard phosphate bufferedsaline solution (PBS) free of Mg⁺⁺ and Ca⁼⁺. The fusion protein isfurther purified by fractionating the resuspended pellet in a denaturingSDS polyacrylamide gel (Sambrook et al., supra). The gel is soaked in0.4 M KCl to visualize the protein, which is excised and electroelutedin gel-running buffer lacking SDS. If the GST/mutant human factor IXfusion protein is produced in bacteria as a soluble protein, it may bepurified using the GST Purification Module (Pharmacia Biotech).

[0066] The fusion protein may be subjected to digestion to cleave theGST from the mature mutant human factor IX protein. The digestionreaction (20-40 μg fusion protein, 20-30 units human thrombin [4000 U/mg(Sigma) in 0.5 mL PBS] is incubated 16-48 hrs, at room temperature andloaded on a denaturing SDS-PAGE gel to fractionate the reactionproducts. The gel is soaked in 0.4 M KCl to visualize the protein bands.The identity of the protein band corresponding to the expected molecularweight of mutant human factor IX may be confirmed by partial amino acidsequence analysis using an automated sequencer (Applied Biosystems Model473A. Foster City, Calif.).

[0067] Alternatively, the DNA sequence encoding the predicted maturemutant human factor IX protein may be cloned into a plasmid containing adesired promoter and, optionally, a leader sequence, see, for example,[Better et al., Science, 240: 104143 (1988)]. The sequence of thisconstruct may be confirmed by automated sequencing. The plasmid is thentransformed into E. coli strain MC1061 using standard proceduresemploying CaCl₂ incubation and heat shock treatment of the bacteria(Sambrook et al., supra). The transformed bacteria are grown in LBmedium supplemented with carbenicillin, and production of the expressedprotein is induced by growth in a suitable medium. If present, theleader sequence will affect secretion of the mature mutant human factorIX protein and be cleaved during secretion.

[0068] The secreted recombinant protein is purified from the bacterialculture media by the method described herein below.

[0069] Mammalian host systems for the expression of the recombinantprotein also are well known to those of skill in the art. Host cellstrains may be chosen for a particular ability to process the expressedprotein or produce certain post-translation modifications that will beuseful in providing protein activity. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing, which cleaves a “prepro” form of theprotein, may also be important to correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38, andthe like have specific cellular machinery and characteristic mechanismsfor such post-translational activities and may be chosen to ensure thecorrect modification and processing of the introduced, foreign protein.

[0070] In a particularly preferred method of recombinant expression ofthe mutant human factor IX proteins of the present invention 293 cellsare co-transfected with plasmids containing the mutant human factor IXcDNA in the pCMN⁷ vector (5′ CMV promoter, 3′ HGH poly A sequence) andpSV2neo (containing the neo resistance gene) by the calcium phosphatemethod. Preferably, the vectors should be linearized with ScaI prior totransfection. Similarly an alternative construct using a similar pCMVvector with the neo gene incorporated can be used. Stable cell lines areselected from single cell clones by limiting dilution in growth mediacontaining 0.5 mg/mL G418 (neomycin like antibiotic) for 10-14 days.Cell lines are screened for mutant factor IX expression by ELISA orWestern blot, and high expressing cell lines are expanded for largescale growth.

[0071] It is preferable that the transformed cells are used forlong-term, high-yield protein production and as such stable expressionis desirable. Once such cells are transformed with vectors that containselectable markers along with the desired expression cassette, the cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The selectable marker is designed to conferresistance to selection and its presence allows growth and recovery ofcells that successfully express the introduced sequences. Resistantclumps of stably transformed cells can be proliferated using tissueculture techniques appropriate to the cell.

[0072] A number of selection systems may be used to recover the cellsthat have been transformed for recombinant protein production. Suchselection systems include, but are not limited to, HSV thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to methotrexate; gpt,that confers resistance to mycophenolic acid; neo, that confersresistance to the aminoglycoside G418; also that confers resistance tochlorsulfuron; and hygro, that confers resistance to hygromycin.Additional selectable genes that may be useful include trpB, whichallows cells to utilize indole in place of tryptophan, or hisD, whichallows cells to utilize histinol in place of histidine. Markers thatgive a visual indication for identification of transformants includeanthocyanins, β-glucuronidase and its substrate, GUS, and luciferase andits substrate, luciferin.

[0073] D. Protein Purification

[0074] It will be desirable to purify the mutant factor IX proteinsgenerated by the present invention. Protein purification techniques arewell known to those of skill in the art. These techniques involve, atone level, the crude fractionation of the cellular milieu to polypeptideand non-polypeptide fractions. Having separated the polypeptide fromother proteins, the polypeptide of interest may be further purifiedusing chromatographic and electrophoretic techniques to achieve partialor complete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography; polyacrylamidegel electrophoresis; and isoelectric focusing. A particularly efficientmethod of purifying peptides is fast protein liquid chromatography oreven BPLC.

[0075] Certain aspects of the present invention concern thepurification, and in particular embodiments, the substantialpurification, of an encoded protein or peptide. The term “purifiedprotein or peptide” as used herein, is intended to refer to acomposition, isolatable from other components, wherein the protein orpeptide is purified to any degree relative to its naturally-obtainablestate. A purified protein or peptide therefore also refers to a proteinor peptide, free from the environment in which it may naturally occur.

[0076] Generally, “purified” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

[0077] Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a f-action by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

[0078] Various techniques suitable for use in protein purification willbe well known to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

[0079] There is no general requirement that the protein or peptidealways be provided in their most purified state. Indeed, it iscontemplated that less substantially purified products will have utilityin certain embodiments. Partial purification may be accomplished byusing fewer purification steps in combination, or by utilizing differentforms of the same general purification scheme. For example, it isappreciated that a cation-exchange column chromatography performedutilizing an HPLC apparatus will generally result in a greater “-fold”purification than the same technique utilizing a low pressurechromatography system. Methods exhibiting a lower degree of relativepurification may have advantages in total recovery of protein product,or in maintaining the activity of an expressed protein.

[0080] It is known that the migration of a polypeptide can vary,sometimes significantly, with different conditions of SDS/PAGE [Capaldiet al., Biochem Biophys Res Comm, 76: 425 (1977)]. It will therefore beappreciated that under differing electrophoresis conditions, theapparent molecular weights of purified or partially purified expressionproducts may vary.

[0081] In particular, the present invention incorporates herein byreference U.S. Pat. Nos. 6,063,909; 6,034,222; 5,639,857 (eachincorporated herein by reference). These documents describe specificexemplary methods for the isolation and purification of factor IXcompositions that may be useful in isolating and purifying the mutanthuman factor IX of the present invention. Given the disclosure of thesepatents, it is evident that one of skill in the art would be well awareof numerous purification techniques that may be used to purify factor IXfrom a given source.

[0082] U.S. Pat. No. 6,063,909 provides methods and compositions forprotecting blood coagulation factor IX from proteases duringpurification or storage. Such methods employ high concentrations of oneor more water soluble organic or inorganic salts to stabilize factor IXagainst conversion to clinically unacceptable peptide structures such asfactor IXa, and/or degraded factor IX peptides. The technique is usefulin stabilizing intermediate purity factor IX preparations duringpurification, and in maintaining the integrity of purified factor IXduring long term storage. One of skill in the art may use methods suchas those disclosed in U.S. Pat. No. 6,063,909 in combination with theinstant invention to provide additional stability to the factor IXpreparations of the present invention.

[0083] U.S. Pat. No. 6,034,222 describes a method for thechromatographic separation of recombinant pro-factor IX from recombinantfactor IX, which employs ion exchangers such as QAE (QAE-Sephadex®, astrong basic anion exchanger comprised of dextran gels that are modifiedby introduction of N,N-diethyl-N-(2-hydroxy-1-propyl)-ammonio-ethylgroups), DEAE (DEAE cellulose, diethylaminoethyl cellulose, anionexchanger) or TMAE (TMAE cellulose, triethylammonioethyl cellulose) andsubsequent elution of factor IX by buffer solutions with high saltconcentrations and/or low pH values.

[0084] Yet another method for the purification of mutant factor IXcontemplates the use of immunoaffinity chromatography using animmunoadsorbent comprising a monoclonal antibody. See, for example,[Liebman et al. Blood, 62(5), supp. 1, 288a (1983); Liebman et al., ProcNat'l Acad Sci USA, 82: 3879-3883 (1985); Bessos, Thrombosis andHaemostasis, 56(1): 86-89 (1986)]. U.S. Pat. No. 5,614,500 describes animmunoaffinity purification of factor IX conducted in the presence of achelating agent. The techniques described therein may be useful in thepresent invention.

[0085] Also it is contemplated that a combination of anion exchange andimmunoaffinity chromatography may be employed to produce purified mutantfactor IX compositions of the present invention.

[0086] In a particularly preferred protocol for protein purification,serum free media containing 10 μg/mL Vitamin K is incubated with aconfluent cell line expressing the mutant human factor IX protein, andharvested every 48 hrs for 10 days. Benzamidine (5 mM) is added, themedia centrifuged at 1200 g to eliminate cellular and particulatedebris, and the conditioned media frozen at −25° C. Upon thawing, theconditioned media is pooled, filtered, and subjected to barium chlorideprecipitation [Cote et al., J Biol Chem, 269(15): 11374-80 (1994)]. Theprecipitate is dissolved in 0.2 M EDTA, and the eluate dialyzedovernight before application to a Mono Q HR 5/5 column(0.15 M NaCl, 20mM HEPES, pH 7.4, 0.1% PEG-8000). Human factor IX is eluted with acalcium chloride gradient (0-45 mM) and concentrated in a Centricon-30.This approach selects for fully gamma-carboxylated factor IX based onthe specificity of the calcium chloride elution. Since this purificationtakes advantage of the unique properties of the Gla domain, mutationsintroduced into the protease domain are not expected to affectpurification of the proteins.

[0087] E. Vectors for Cloning, Gene Transfer and Expression.

[0088] As discussed in the previous section, expression vectors areemployed to express the mutant human factor IX polypeptide product,which can then be purified and used in replacement therapy for thetreatment of hemophilia B. In other embodiments, expression vectors maybe used in gene therapy applications to introduce the mutant factorIX-encoding nucleic acids into cells in need thereof and/or to inducemutant factor IX expression in such cells. The present section isdirected to a description of the production of such expression vectors.

[0089] Expression requires that appropriate signals be provided in thevectors, and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare described. The conditions for the use of a number of dominant drugselection markers for establishing permanent, stable cell clonesexpressing the products also are provided, as is an element that linksexpression of the drug selection markers to expression of thepolypeptide.

[0090] a. Regulatory Elements.

[0091] Promoters and Enhancers. Throughout this application, the term“expression construct” or “expression vector” is meant to include anytype of genetic construct containing a nucleic acid coding for geneproducts in which part or all of the nucleic acid encoding sequence iscapable of being transcribed. The transcript may be translated into aprotein, but it need not be In certain embodiments, expression includesboth transcription of a gene and translation of mRNA into a geneproduct. The nucleic acid encoding a gene product is undertranscriptional control of a promoter. A “promoter” refers to a DNAsequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. The phrase “under transcriptional control”means that the promoter is in the correct location and orientation inrelation to the nucleic acid to control RNA polymerase initiation andexpression of the gene.

[0092] The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

[0093] At least one module in each promoter functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as the promoter forthe mammalian terminal deoxynucleotidyl transferase gene and thepromoter for the SV40 late genes, a discrete element overlying the startsite itself helps to fix the place of initiation.

[0094] Additional promoter elements regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the tk promoter, thespacing between promoter elements can be increased to 50 bp apart beforeactivity begins to decline. Depending on the promoter, it appears thatindividual elements can function either co-operatively or independentlyto activate transcription.

[0095] The particular promoter employed to control the expression of anucleic acid sequence of interest is not believed to be important, solong as it is capable of directing the expression of the nucleic acid inthe targeted cell. Thus, where a human cell is targeted, it ispreferable to position the nucleic acid coding region adjacent to andunder the control of a promoter that is capable of being expressed in ahuman cell. Generally speaking, such a promoter might include either ahuman or viral promoter.

[0096] In various embodiments, the human cytomegalovirus (CMV) immediateearly gene promoter, the SV40 early promoter, the Rous sarcoma viruslong terminal repeat, β-actin, rat insulin promoter, the phosphoglycerolkinase promoter and glyceraldehyde-3-phosphate dehydrogenase promoter,all of which are promoters well known and readily available to those ofskill in the art, can be used to obtain high-level expression of thecoding sequence of interest. The use of other viral or mammaliancellular or bacterial phage promoters that are well-known in the art toachieve expression of a coding sequence of interest is contemplated aswell, provided that the levels of expression are sufficient for a givenpurpose. By employing a promoter with well known properties, the leveland pattern of expression of the protein of interest followingtransfection or transformation can be optimized.

[0097] Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product. Several inducible promoter systems are available forproduction of viral vectors. One such system is the ecdysone system(Invitrogen, Carlsbad. Calif.), which is designed to allow regulatedexpression of a gene of interest in mammalian cells. It consists of atightly regulated expression mechanism that allows virtually no basallevel expression of the transgene, but over 200-fold inducibility.

[0098] Another inducible system that would be useful is the Tet-Off™ orTet-On™ system (Clontech, Palo Alto, Calif.) originally developed byGossen and Bujard [Proc Nat'l Acad Sci USA, 15;89(12):5547-51 (1992);Gossen et al., Science, 268(5218): 1766-69 (1995)].

[0099] In some circumstances, it may be desirable to regulate expressionof a transgene in a gene therapy vector. For example, different viralpromoters with varying strengths of activity may be utilized dependingon the level of expression desired. In mammalian cells, the CMVimmediate early promoter is often used to provide strong transcriptionalactivation. Modified versions of the CMV promoter that are less potenthave also been used when reduced levels of expression of the transgeneare desired. When expression of a transgene in hematopoetic cells isdesired, retroviral promoters such as the LTRs from MLV or MMTV areoften used. Other viral promoters that may be used depending on thedesired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenoviruspromoters such as from the E1A, E2A, or MLP region, AAV LTR, cauliflowermosaic virus, HSV-TK, and avian sarcoma virus.

[0100] Similarly, tissue specific promoters may be used to affecttranscription in specific tissues or cells to reduce potential toxicityor undesirable effects on non-targeted tissues. For example, promoterssuch as the PSA, probasin, prostatic acid phosphatase, orprostate-specific glandular kallikrein (hK2) may be used to target geneexpression in the prostate.

[0101] In certain indications, it may be desirable to activatetranscription at specific times after administration of the gene therapyvector. This may be done with such promoters as those that are hormoneor cytokine regulatable. For example, in gene therapy applications wherethe indication is a gonadal tissue where specific steroids are producedor routed to, use of androgen or estrogen regulated promoters may beadvantageous. Such promoters that are hormone regulatable include MMTV.MT-1, ecdysone, and RuBisco. Other hormone regulated promoters such asthose responsive to thyroid, pituitary, and adrenal hormones areexpected to be useful in the present invention. Cytokine andinflammatory protein responsive promoters that could be used include Kand T Kininogen [Kageyama et al., J Biol Chem, 262(5): 2345-51 (1987)],c-fos, TNF-alpha, C-reactive protein [Arcone et al., Nucl Acids Res16(8): 3195-207 (1988)], haptoglobin [Oliviero et al., EMBO J, 6(7):1905-12 (1987)], serum amyloid A2, C/EBP alpha, IL-1, IL-6 [Poli andCortese, Proc Nat'l Acad Sci USA, 86(21): 8202-6 (1989)], complement C3[Wilson et al., Mol Cell Biol 10(12): 6181-91 (1990)], IL-8, alpha-1acid glycoprotein [Prowse and Baumann, Mol Cell Biol, 8(1): 42-51(1988)], alpha-1 antitypsin, lipoprotein lipase [Zechner et al., MolCell Biol, 8(6): 2394-401 (1988)], angiotensinogen [Ron et al., Mol CellBiol 11(5): 2887-95 (1991)], fibrinogen, c-jun (inducible by phorbolesters, TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide),collagenase (induced by phorbol esters and retinoic acid),metallothionein (heavy metal and glucocorticoid inducible), stromelysin(inducible by phorbol ester, interleukin-1 and EGF), alpha-2macroglobulin, and alpha-1 antichymotrypsin.

[0102] Other promoters that could be used according to the presentinvention include Lac-regulatable, heat (hyperthermia)-induciblepromoters, and radiation-inducible, for e.g., EGR [Joki et al., Hum GeneTher; 6(12): 1507-13 (1995)], alpha-inhibin, RNA pol III tRNA met andother amino acid promoters. U1 snRNA [Bartlett et al., Proc Nat'l AcadSci USA, 20;93(17): 8852-7 (1996)], MC-1, PGK, β-actin, and α-globin.Many other promoters that may be useful are listed in Walther and Stein[J Mol Med 74(7): 379-92 (1996)].

[0103] It is envisioned that any of the above promoters alone, or incombination with another, may be useful according to the presentinvention depending on the action desired. In addition, this list ofpromoters should not be construed to be exhaustive or limiting, andthose of skill in the art will know of other promoters that may be usedin conjunction with the promoters and methods disclosed herein.

[0104] Another regulatory element contemplated for use in the presentinvention is an enhancer. These are genetic elements that increasetranscription from a promoter located at a distant position on the samemolecule of DNA. Enhancers are organized much like promoters. That is,they are composed of many individual elements, each of which binds toone or more transcriptional proteins. The basic distinction betweenenhancers and promoters is operational. An enhancer region as a wholemust be able to stimulate transcription at a distance; this need not betrue of a promoter region or its component elements. On the other hand,a promoter must have one or more elements that direct initiation of RNAsynthesis at a particular site and in a particular orientation, whereasenhancers lack these specificities. Promoters and enhancers are oftenoverlapping and contiguous, often seeming to have a very similar modularorganization. Enhancers useful in the present invention are well knownto those of skill in the art and will depend on the particularexpression system being employed [Scharf et al., Results Probl CellDiffer 20: 125-62 (1994); Bittner et al., Meth Enzymol 153: 516-544(1987)].

[0105] Polyadenylation Signals. Where a cDNA insert is employed, onewill typically desire to include a polyadenylation signal to affectproper polyadenylation of the gene transcript. The nature of thepolyadenylation signal is not believed to be crucial to the successfulpractice of the invention, and any such sequence may be employed, suchas human or bovine growth hormone and SV40 polyadenylation signals. Alsocontemplated as an element of the expression cassette is a terminator.These elements can serve to enhance message levels and to minimizeread-through from the cassette into other sequences.

[0106] IRES. In certain embodiments of the invention, the use ofinternal ribosome entry site (IRES) elements is contemplated to createmultigene, or polycistronic, messages. IRES elements are able to bypassthe ribosome scanning model of 5′ methylated Cap dependent translationand begin translation at internal sites [Pelletier and Sonenberg,Nature, 334: 320-325 (1988)]. IRES elements from two members of thepicomavirus family (poliovirus and encephalomyocarditis) have beendescribed [Pelletier and Sonenberg (1988), supra], as well an IRES froma mammalian message [Macejak and Sarnow, Nature, 353: 90-94 (1991)].IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message.

[0107] Any heterologous open reading frame can be linked to IRESelements. This includes genes for secreted proteins, multi-subunitproteins, encoded by independent genes, intracellular or membrane-boundproteins and selectable markers. In this way, expression of severalproteins can be simultaneously engineered into a cell with a singleconstruct and a single selectable marker.

[0108] b. Delivery of Expression Vectors.

[0109] There are a number of ways in which expression vectors mayintroduced into cells. In certain embodiments of the invention, theexpression construct comprises a virus or engineered construct derivedfrom a viral genome. In other embodiments, non-viral delivery iscontemplated. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells[Ridgeway, In: Rodriguez R L, Denhardt D T, ed. Vectors: A survey ofmolecular cloning vectors and their uses. Stoneham: Butterworth,467-492, 1988; Nicolas and Rubenstein, In: Vectors: A survey ofmolecular cloning vectors and their uses, Rodriguez & Denhardt (eds.),Stoneham: Butterworth, 493-513, 1988; Baichwal and Sugden, In: GeneTransfer, Kucherlapati R, ed., New York, Plenum Press, 117-148, 1986;Temin, In: gene Transfer, Kucherlapati (ed.), New York: Plenum Press,149-188, 1986]. The first viruses used as gene vectors were DNA virusesincluding the papovaviruses (simian virus 40, bovine papilloma virus,and polyoma) [Ridgeway, (1988), supra; Baichwal and Sugden, (1986),supra] and adenoviruses [Ridgeway, (1988), supra; Baichwal and Sugden,(1986), supra]. These have a relatively low capacity for foreign DNAsequences and have a restricted host spectrum. Furthermore, theironcogenic potential and cytopathic effects in permissive cells raisesafety concerns. They can accommodate only up to 8 kb of foreign geneticmaterial but can be readily introduced in a variety of cell lines andlaboratory animals [Nicolas and Rubenstein, (1988), supra; Temin,(1986), supra].

[0110] It is now widely recognized that DNA may be introduced into acell using a variety of viral vectors. In such embodiments, expressionconstructs comprising viral vectors containing the genes of interest maybe adenoviral (see, for example, U.S. Pat. Nos. 5,824,544; 5,707,618;5,693,509; 5,670,488; 5,585,362; each incorporated herein by reference),retroviral (see, for example, U.S. Pat. Nos. 5,888,502; 5,830,725;5,770,414; 5,686,278; 4,861,719 each incorporated herein bad reference),adeno-associated viral (see, for example, U.S. Pat. Nos. 5,474,935;5,139,941; 5,622,856; 5,658,776; 5,773,289; 5,789,390; 5,834,441;5,863,541; 5,851,521; 5,252,479 each incorporated herein by reference),an adenoviral-adenoassociated viral hybrid (see, for example, U.S. Pat.No. 5,856,152 incorporated herein by reference) or a vaccinia viral or aherpesviral (see, for example, U.S. Pat. Nos. 5,879,934; 5,849,571;5,830,727; 5,661,033; 5,328,688 each incorporated herein by reference)vector.

[0111] There are a number of alternatives to viral transfer of geneticconstructs. This section provides a discussion of methods andcompositions of non-viral gene transfer. DNA constructs of the presentinvention are generally delivered to a cell, and in certain situations,the nucleic acid or the protein to be transferred may be transferredusing non-viral methods.

[0112] Several non-viral methods for the transfer of expressionconstructs into cultured mammalian cells are contemplated by the presentinvention. These include calcium phosphate precipitation [Graham and VanDer Eb, Virology, 52: 456-467 (1973); Chen and Okayama, Mol Cell Biol,7: 2745-2752 (1987); Rippe et al., Mol Cell Biol, 10: 689-695 (1990)]DEAE-dextran [Gopal, Mol Cell Biol, 5: 1188-1190 (1985)],electroporation [Tur-Kaspa et al., Mol Cell Biol, 6: 716-718 (1986).Potter et al., Proc Nat'l Acad Sci USA, 81: 7161-7165 (1984)], directmicroinjection [Harland and Weintraub, J Cell Biol, 101: 1094-1099(1985)], DNA-loaded liposomes [Nicolau and Sene, Biochim Biophys Acta,721: 185-190 (1982); Fraley et al., Proc Nat'l Acad Sci USA, 76:3348-3352 (1979); Felgner, Sci Amer 276(6): 102-6 (1997); Feigner, HumGene Ther 7(15): 1791-3 (1996)], cell sonication [Fechheimer et al.,Proc Nat'l Acad Sci USA, 84: 8463-8467 (1987)], gene bombardment usinghigh velocity mnicroprojectiles [Yang et al., Proc Nat'l Acad Sci USA,87: 9568-9572 (1990)], and receptor-mediated transfection [Wu and Wu, JBiol Chem, 262: 4429-4432 (1987); Wu and Wu, Biochemistry, 27: 887-892(1988); Wu and Wu, Adv Drug Deliv Rev, 12: 159-167 (1993)].

[0113] Once the construct has been delivered into the cell, the nucleicacid encoding the therapeutic gene may be positioned and expressed atdifferent sites. In certain embodiments, the nucleic acid encoding thetherapeutic gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell, and where inthe cell the nucleic acid remains, is dependent on the type ofexpression construct employed.

[0114] In a particular embodiment of the invention, the expressionconstruct may be entrapped in a liposome. Liposomes are vesicularstructures characterized by a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers [Ghosh andBachhawat, In: Liver diseases, targeted diagnosis and therapy usingspecific receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker,pp. 87-104. (1991)]. The addition of DNA to cationic liposomes causes atopological transition from liposomes to optically birefringentliquid-crystalline condensed globules [Radler et al., Science,275(5301): 810-4 (1997)]. These DNA-lipid complexes are potentialnon-viral vectors for use in gene therapy and delivery.

[0115] Liposome-mediated nucleic acid delivery and expression of foreignDNA in vitro has been very successful. Also contemplated in the presentinvention are various commercial approaches involving “lipofection”technology. In certain embodiments of the invention, the liposome may becomplexed with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA [Kaneda et al., Science, 243: 375-378 (1989)].In other embodiments, the liposome maybe complexed or employed inconjunction with nuclear nonhistone chromosomal proteins (HMG-1) [Katoet al., J Biol Chem, 266: 3361-3364 (1991)]. In yet further embodiments,the liposome may be complexed or employed in conjunction with both HVJand HMG-1. In that such expression constructs have been successfullyemployed in transfer and expression of nucleic acid in vitro and invivo, then they are applicable for the present invention.

[0116] Other vector delivery systems that can be employed to deliver anucleic acid encoding a therapeutic gene into cells includereceptor-mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis inalmost all eukaryotic cells. Because of the cell type-specificdistribution of various receptors, the delivery can be highly specific[Wu and Wu, (1993), supra].

[0117] Receptor-mediated gene targeting vehicles generally consist oftwo components: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) [Wuand Wu, (1987), supra] and transferrin [Wagner et al., Proc Nat'l AcadSci USA, 87(9): 3410-3414 (1990)]. Recently, a syntheticneoglycoprotein, which recognizes the same receptor as ASOR, has beenused as a gene delivery vehicle [Ferkol et al., FASEB J. 7: 1081-1091(1993); Perales et al., Proc Nat'l Acad Sci USA, 91: 4086-4090 (1994)]and epidermal growth factor (EGF) has also been used to deliver genes tosquamous carcinoma cells (Myers, EPO 0273085).

[0118] In other embodiments, the delivery vehicle may comprise a ligandand a liposome. For example, Nicolau et al. [Meth Enzymol, 149: 157-176(1987)] employed lactosyl-ceramide, a galactose-terminalasialganglioside, incorporated into liposomes and observed an increasein the uptake of the insulin gene by hepatocytes. Thus, it is feasiblethat a nucleic, acid encoding a therapeutic gene also may bespecifically delivered into a particular cell type by any number ofreceptor-ligand systems with or without liposomes.

[0119] In another embodiment of the invention, the expression constructmay simply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be preformed by any of the methods mentioned above thatphysically or chemically permeabilize the cell membrane. This isapplicable particularly for transfer in vitro, however, it may beapplied for in vivo use as well. Dubensky et al. [Proc Nat'l Acad SciUSA, 81: 7529-7533 (1984)] successfully injected polyomavirus DNA in theform of CaPO₄ precipitates into liver and spleen of adult and newbornmice demonstrating active viral replication and acute infection.Benvenisty and Neshif [Proc Nat'l Acad Sci USA, 83:9551-9555 (1986)]also demonstrated that direct intraperitoneal injection of CaPO₄precipitated plasmids results in expression of the transfected genes.

[0120] Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them [Klein et al., Nature, 327: 70-73 (1987)].Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force [Yang etal., Proc Nat'l Acad Sci USA, 87: 9568-9572 (1990)]. Themicroprojectiles used have consisted of biologically inert substancessuch as tungsten or gold beads.

[0121] F. Methods of Treating Hemophilia B.

[0122] As mentioned herein above, it is contemplated that the mutanthuman factor IX protein or the vectors comprising a polynucleotideencoding such a protein will be employed in replacement therapyprotocols for the treatment of hemophilia B.

[0123] a. Protein Based Therapy

[0124] One of the therapeutic embodiments of the present invention isthe provision, to a subject in need thereof, compositions comprising themutant human factor IX protein of the present invention. As discussedabove, the protein may have been generated through recombinant means orby automated peptide synthesis. The factor IX formulations for such atherapy may be selected based on the route of administration and mayinclude liposomal formulations as well as classic pharmaceuticalpreparations.

[0125] The mutant human factor IX proteins are formulated intoappropriate preparation and administered to one or more sites within thesubject in a therapeutically effective amount. In particularly preferredembodiments, the mutant human factor IX protein based therapy iseffected via continuous or intermittent intravenous administration. By“therapeutically effective amount” the present invention refers to thatamount of mutant human factor IX that is sufficient to produce orenhance the coagulation of blood in a mammal following a bleed. Forexample, a therapeutically effective amount may enhance coagulation byreducing clotting times in a blood coagulation assay, or even increaseformation of intrinsic tenase or factor X activation. Blood coagulationassays are well known to those of skill in the art and are described forexample, in Walter et al., [Proc Nat'l Acad Sci USA, 93: 3056-3061(1996); Hathaway and Goodnight (1993), Laboratory Measurement ofHemostasis and Thrombosis, In: Disorders of Hemostasis and Thrombosis: AClinical Guide, pp. 21-29)].

[0126] Those of skill in the art will understand that the amounts ofmutant human factor IX for therapeutic use may vary. It is contemplatedthat the specific activity of the factor IX protein preparation may bein the range of from about 100 units/mg of protein to about 500 units/mgprotein. Thus, a given preparation of mutant human factor IX maycomprises about 100 units/mg protein, about 125 units/mg protein, about150 units/mg protein, about 175 units/mg protein, about 200 units/mgprotein, about 225 units/mg protein, about 250 units/mg protein, about275 units/mg protein, about 300 units/mg protein, about 325 units/mgprotein, about 350 units/mg protein, about 375 units/mg protein, about400 units,/mg protein, about 425 units/mg protein, about 450 units/mgprotein, about 475 units/mg protein and about 500 units/mg protein. Aparticularly preferred range is from about 100 units/mg protein to about200 units/mg protein, a more preferable range is between about 150 toabout 200 units/mg protein. Preferably, the protein composition issubstantially free of contaminating factor IXa and has a factor IXacontamination level of less than 0.02% (w/w). Factor IX compositions,suitable for injection into a patient, can be prepared for example, byreconstitution with a pharmacologically acceptable diluent of alyophilized sample comprising purified factor IX and stabilizing salts.

[0127] Administration of the compositions can be systemic or local andmay comprise a single-site injection of a therapeutically effectiveamount of the mutant human factor IX protein composition. Any routeknown to those of skill in the art for the administration of atherapeutic composition of the invention is contemplated including forexample, intravenous, intramuscular, subcutaneous, or a catheter forlong-term administration. Alternatively, it is contemplated that thetherapeutic composition may be delivered to the patient at multiplesites. The multiple administrations may be rendered simultaneously ormay be administered over a period of several hours. In certain cases itmay be beneficial to provide a continuous flow of the therapeuticcomposition. Additional therapy may be administered on a periodic basis,for example, daily, weekly, or monthly.

[0128] b. Genetic Based Therapies.

[0129] Another therapeutic embodiment contemplated by the presentinvention is a method of treating a mammal having hemophilia comprisingadministering to the mammal a gene therapy based pharmaceuticalcomposition. Specifically, the present inventors intend to provide, to agiven tissue in a patient or subject in need thereof, an expressionconstruct capable of providing the mutant human factor IX to thatpatient in a functional form. It is specifically contemplated that agene encoding the mutant human factor IX will be employed in humantherapy. The lengthy discussion of expression vectors and the geneticelements employed therein is incorporated into this section byreference. Particularly preferred expression vectors are viral vectorssuch as adenovirus, adeno-associated virus, herpesvirus, vaccinia virus,and retrovirus. Also preferred is a liposomally-encapsulated expressionvector.

[0130] Those of skill in the art are well aware of how to apply genedelivery in vivo. For viral vectors, one generally will prepare a viralvector stock. Depending on the kind of virus and the titer attainable,one will deliver 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰,1×10¹¹ or 1×10¹² infectious particles to the patient. Similar figuresmay be extrapolated for liposomal or other non-viral formulations bycomparing relative uptake efficiencies. Formulation as apharmaceutically acceptable composition is discussed below.

[0131] Various routes are contemplated for delivery. The section belowon routes contains an extensive list of possible routes. For example,systemic delivery is contemplated. In other cases, a variety of direct,local and regional approaches may be taken. For example, where theindividual being treated exhibits a localized bleed, that area may bedirectly injected with the expression vector.

[0132] In certain embodiments, it is contemplated that a preparation ofthe vector comprising the mutant human factor IX encoding polynucleotideis injected into the muscle tissue of an animal at a single site perdose. In other embodiments, the preparation is injected into the muscletissue of the animal either simultaneously, or over the course ofseveral hours, at multiple muscle tissue sites. In the latter instance,when the method comprises simultaneous multiple injections of viralvector genomes, it is envisaged that a multiple delivery injectiondevice may be used such that different areas of muscle tissue receivethe vector simultaneously.

[0133] Incorporated herein by reference is U.S. Pat. No. 6,093,392 thatdescribes methods of gene therapy for hemophilia, which employadeno-associated viral vectors. Similarly, U.S. Pat. No. 5,935,935 isincorporated herein by reference and describes the use of adenoviralvectors for the treatment of hemophilia. It is contemplated that themethods described therein will be useful in combination with thecompositions of the present invention.

[0134] Also incorporated herein by reference is U.S. Pat. No. 5,681,746,which describes retroviral vectors for the expression of factor VIII andpharmaceutical compositions and methods of using such vectors fortreating hemophilia. The present invention contemplates gene therapyprotocols in which such retroviral particles comprising mutant humanfactor IX compositions of the present invention may be used for thetreatment of mammals afflicted with hemophilia

[0135] C. Combination Therapy

[0136] In addition to therapies based solely on the delivery of themutant human factor IX, combination therapy is specificallycontemplated. In the context of the present invention, it iscontemplated that the mutant human factor IX therapy could be usedsimilarly in conjunction with other agents for commonly used for thetreatment of hemophilia.

[0137] To achieve the appropriate therapeutic outcome, using the methodsand compositions of the present invention, one would generally provide acomposition comprising the mutant human factor IX and at least one othertherapeutic agent (second therapeutic agent). In the present invention,it is contemplated that the second therapeutic agent may be one or moreother factors involved in the blood coagulation cascade. For example, itis contemplated that the compositions comprising the mutant human factorIX of the present invention may be combined with activated prothrombincomplex concentrates, factors II, VII, VIIa, VIII, X, precursor Xa,protein C, XI and XII.

[0138] The combination therapy compositions would be provided in acombined amount effective to produce the desired therapeutic outcome ofblood coagulation. This process may in-solve contacting the cells withthe mutant human factor IX and the second agent(s) or factor(s) at thesame time. This may be achieved by administering a single composition orpharmacological formulation that includes both agents, or byadministering two distinct compositions or formulations, at the sametime, wherein one composition includes the mutant human factor IXtherapeutic composition and the other includes the second therapeuticagent.

[0139] Alternatively, the mutant human factor IX treatment may precedeor follow the other agent treatment by intervals ranging from minutes toweeks. In embodiments where the second therapeutic agent and the mutanthuman factor IX are administered separately, one would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the second agent and mutant human factor IXwould still be able to exert an advantageously combined effect. In suchinstances, it is contemplated that one would administer both modalitieswithin about 12-24 hours of each other and, more preferably, withinabout 6-12 hours of each other, with a delay time of only about 12 hoursbeing most preferred. In some situations, it may be desirable to extendthe time period for treatment significantly, however, where several days(2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations.

[0140] Local delivery of mutant human factor IX expression constructs orproteins to patients may be a very efficient method for delivering atherapeutically effective gene to counteract the immediate clinicalmanifestations of the disease, i.e., localized bleeding. Similarly, thesecond therapeutic agent may be directed to a particular, affectedregions of the subject's body. Alternatively, systemic delivery of themutant human factor IX and/or the second therapeutic agent may beappropriate in certain circumstances.

[0141] G. Assays for Factor IX activity

[0142] In certain aspects of the present invention, it may be necessaryto determine the activity of mutant human factor A. In particular, theeffect of the therapeutic compositions of the present invention on bloodcoagulation activity may need to be monitored Those of skill in the artare aware of numerous blood coagulation assays some of which aredescribed in the present section. This is by no means intended to be anexhaustive list of such assays and is merely intended to provide certainexemplary assays well known to those of skill in the art that may beused in determining the blood coagulation activity of the presentinvention. Further, the present section also describes assays for thedetermination of heparin inhibition of factor IX activity. Exemplary invitro and in vivo assays for determining these activities are providedherein below.

[0143] a. In vitro assays

[0144] A chromogenic in vitro assay may be used to determine the humanfactor IX activity of a given mutant human factor IX of the presentinvention. Factor IXa has poor reactivity toward chromogenic substrates,likely due to the partially collapsed nature of the active site[Brandstetter et al., Proc Nat'l Acad Sci USA, 92(21): 9796-800 (1995)].However, the addition of 30% ethylene glycol can increase the catalyticrate nearly ten fold, especially for substrates with hydrophobicmoieties in the P3 position [Sturzebecher et al., FEBS Letters, 412(2):295-300 (1997)]. The effect of mutations in the heparin binding site offactor IXa on cleavage of the chromogenic substrates, Pefachrome IXa(CH₃SO₂-D-CHG-Gly-Arg-pNA) or CBS 31.39(CH₃SO₂DLeu-Gly-Arg-p-nitroanilide), can be assessed by incubatingincreasing amounts of heparin with 25 nM enzyme in 0.15 M NaCl, 2 nMCaCl₂, 20 mM HEPES, pH 7.4, 30% ethylene glycol and 2.5 mM PefachromeIXa or 4 mM CBS 31.39 in a microtiter plate. Initial rates aredetermined by the change in absorbance at 405 nm over 5-10 min a VmaxReader. The K_(m) of CBS 31.39 for factor IXa is 3.7 mM under theseconditions.

[0145] An in vitro assay for intrinsic complex activity to determinefactor IX activity also may be used. In this assay, thrombin-activatedfactor VIIIa (final concentration 0.5 nM) is added to a reactioncontaining 5 nM factor IXa, 5% (v/v) rabbit brain cephalin, 300 nMfactor X, and increasing concentrations of heparin in 0.15 M NaCl, 20 nMHEPES, pH 7.4, 2 mM CaCl2, and 0.1% PEG-8000. The reaction are sampled(50 μl) at 15, 30, 45, and 60 sec into 10 μl of 0.25 M EDTA, pH 8.0. Thechromogenic substrate S-2765 is then added at 300 μM and the amount offactor Xa generated determined by comparison of the rate of cleavagewith a standard curve. The assay for intrinsic tenase activity may bemodified to be performed in the presence of excess factor VIIIa (5 nM)and the linear range for factor IXa determined (as previously describedfor factor VIIIa) for accurate quantitation of the mutant activities.Significant differences in catalytic activity may be further analyzed bydetermination of the Km and kcat for factor X activation by intrinsictenase for wild-type and mutant factor IXa. The affinity of mutantfactor IXa-factor VIIIa complex formation in the presence ofphospholipid can be compared to wild-type factor IXa in a kineticbinding assay.

[0146] The relative affinity of heparin for the mutant human factor IXaproteins can be determined by titration of active site-labeled protease.The interaction of heparin with F1-EGR-factor IXa can be detected by thechange in emission fluorescence intensity at 525 nm. To generate theactive site-labeled proteases, wild-type and mutant human factor IX isactivated by incubation with factor XIa. Conditions for completeactivation can be confirmed by SDS-PAGE for each mutant protein. Themutant factor IXa is then incubated with ten-fold molar excess offluorescein-EGR-chloromethylketone (Hematologic Technologies) for 30 minat 23° C., followed by gel filtration chromatography on a G-100 column(fractionation range 4-100 kDa) to separate factor XIa (void volume) andthe low molecular weight free inhibitor from F1-EGR-factor IXa. Thesample may then be subjected to additional dialysis if necessary tocompletely remove free inhibitor. Labeled proteases will then bequantitated by A₂₈₀. The FI-EGR-factor IXa (25 nM) is titrated withsize-fractionated heparin chains to generate binding curves. The bindingcurves for mutant factor IXa can be compared to wild-type underidentical conditions, with fitting to an appropriate site-specificbinding model to provide a KD(obs) [Olson et al., J Biol Chem, 266(10):6342-52 (1991)]. An estimate of the relative affinity of mutant humanfactor IXa for heparin (i.e. rank order) is sufficient to correlate withthe relative effect of mutations on enzymatic activity and inhibition byheparin. This strategy is similar to that used to map the heparinbinding site of thrombin, where NaCl elution from heparin-sepharose wasused as an estimate of heparin affinity, allowing correlation of elutionposition with the rate constant for inhibition by ATIII-heparin [Sheehanand Sadler, Proc Nat'l Acad Sci USA, 91(12): 5518-22 (1994)].

[0147] In vitro blood coagulation assays also are well known to those ofskill in the art and are described, for example, in Walter et al., [ProcNat'l Acad Sci USA, 93: 3056-3061 (1996); Hathaway and Goodnight (1993),Laboratory Measurement of Hemostasis and Thrombosis, In: Disorders ofHemostasis and Thrombosis: A Clinical Guide, pp. 21-29)]. These assaysmay be used in the present invention to ensure that the mutant humanfactor IX possesses an appropriate blood coagulation effect. Those ofskill in the art also are referred to “A Laboratory Manual of BloodCoagulation” Austen et al., Blackwell Scientific Publishing (1975) foradditional methods for conducting blood clotting assays.

[0148] In preferred embodiments, the effect of mutations in the heparinbinding exosite on the coagulant activity of mutant human factor IX isassessed by performing an activated partial thromboplastin time (APTT)in factor IX deficient plasma [Bajaj et al, Meth Enzymol, 222: 96-128(1993)]. The relative coagulant activity of the mutants is determined bycomparison to a standard curve. The APTT reflects both activation of themutant human factor IX by factor XIa, and the enzymatic activity of theprotease in plasma. Unexpected differences can be further analyzed bycomparing mutant human factor IX and factor IXa plasma coagulantactivity of the mutants to wild-type, in order to differentiate effectson activation versus enzymatic activity.

[0149] b. In Vivo Assays

[0150] Before the mutant human factor IX compositions of the presentinvention are employed in human therapeutic protocols, it may bedesirable to monitor the effects of such compositions in animal models.There are a number of animal models, in vivo assays, previouslydescribed by those of skill in the art that may be useful in the presentinvention.

[0151] An exemplary animal model for hemophilia B is available. Forexample, a colony of mice having severe hemophilia B are well known tothose of skill in the art [Lin et al., Blood, 90(10): 3962-6 (1997);Kung et al., Blood, 91(3): 784-90 (1998); Snyder et al., Nat Med, 5(1):64-70 (1999)]. Additionally, a colony of dogs having severe hemophilia Bcomprising males that are hemizygous and females that are homozygous fora point mutation in the catalytic domain of the canine factor IX gene,have been maintained for more than two decades at the University ofNorth Carolina, Chapel Hill [Evans et al., Blood 74: 207-212 (1989)].

[0152] The hemostatic parameters of the above mice and dogs are welldescribed. For example, in the dogs there is an absence of plasma factorIX antigen, whole blood clotting times of >60 minutes, whereas normaldogs are 6-8 minutes, and prolonged activated partial thromboplastintime of 50-80 seconds, whereas normal dogs are 13-18 seconds. These dogsexperience recurrent spontaneous hemorrhages. Typically, significantbleeding episodes are successfully managed by the single intravenousinfusion of 10 mL/kg of normal canine plasma; occasionally, repeatinfusions are required to control bleeding.

[0153] In order to determine the efficacy of the mutant human factor IXprotein and gene therapy compositions of the present invention, suchmice and dogs may be injected intramuscularly and/or intravenously withthe compositions of the present invention and the blood clotting time inthe presence and absence of the compositions may be determined. Suchdeterminations will be helpful in providing guidance on the dosages andtimes of administration and the efficacy of a given composition againsthemophilia B. In gene therapy protocols, immunofluorescence staining ofsections obtained from biopsied muscle may be performed, and expressionof the mutant human factor IX in the transduced muscle fibers may bedetermined.

[0154] H. Pharmaceutical Compositions

[0155] In order to prepare mutant human factor IX containingcompositions for clinical use, it will be necessary to prepare the viralexpression vectors, proteins, and nucleic acids as pharmaceuticalcompositions, i.e., in a form appropriate for in vivo applications.Generally, this will entail preparing compositions that are essentiallyfree of pyrogens, as well as other impurities that could be harmful tohumans or animals.

[0156] One will generally desire to employ appropriate salts and buffersto render delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the mutant human factor IX or an expression vectorto cells, dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium. Such compositions also are referred to asinocula. The phrase “pharmaceutically or pharmacologically acceptable”refer to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the vectors or cells of the presentinvention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

[0157] The active compositions of the present invention include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. The pharmaceuticalcompositions may be introduced into the subject by any conventionalmethod, e.g., by intravenous, intradermal, intramusclar, intramammary,intraperitoneal, intrathecal, retrobulbar, intrapulmonary (e.g. termrelease); by oral, sublingual, nasal, anal, vaginal, or transdermaldelivery, or by surgical implantation at a particular site. Thetreatment may consist of a single dose or a plurality of doses over aperiod of time.

[0158] The active compounds may be prepared for administration assolutions of free base or pharmacologically acceptable salts in water,suitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions also can be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof, and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

[0159] The pharmaceutical forms, suitable for injectable use, includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion, and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

[0160] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle that contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum-drying and freeze-drying techniquesthat yield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof

[0161] As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions.

[0162] For oral administration the polypeptides of the present inventionmay be incorporated with excipients and used in the form ofnon-ingestible mouthwashes and dentifrices. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The active ingredient may also be dispersed in dentifrices,including: gels, pastes, powders and slurries. The active ingredient maybe added in a therapeutically effective amount to a paste dentifricethat may include water, binders, abrasives, flavoring agents, foamingagents, and humectants.

[0163] The compositions of the present invention may be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups alsocan be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

[0164] The compositions of the present invention may be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups alsocan be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

[0165] Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms, such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution should be suitably buffered, ifnecessary, and the liquid diluent first rendered isotonic withsufficient saline or glucose. These particular aqueous solutions areespecially suitable for intravenous, intramuscular, subcutaneous andintraperitoneal administration.

[0166] “Unit dose” is defined as a discrete amount of a therapeuticcomposition dispersed in a suitable carrier. For example, wherepolypeptides are being administered parenterally, the polypeptidecompositions are generally injected in doses ranging from 1 μg/kg to 100mg/kg body weight/day, preferably at doses ranging from 0.1 mg/kg toabout 50 mg/kg body weight/day. In terms of units of mutant human factorIX activity per kg of weight of subject, it is contemplated that betweenabout 100 to about 500 units/kg body weight will be useful. Parenteraladministration may be carried out with an initial bolus followed bycontinuous infusion to maintain therapeutic circulating levels of drugproduct. Those of ordinary skill in the art will readily optimizeeffective dosages and administration regimens as determined by goodmedical practice and the clinical condition of the individual patient.

[0167] The frequency of dosing will depend on the pharmacokineticparameters of the agents and the routes of administration. The optimalpharmaceutical formulation will be determined by one of skill in the artdepending on the route of administration and the desired dosage. See,for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990), MackPubl. Co, Easton Pa. 18042, pp. 1435-1712, incorporated herein byreference. Such formulations may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theadministered agents. Depending on the route of administration, asuitable dose may be calculated according to body weight, body surfacearea, or organ size. Further refinement of the calculations necessary todetermine the appropriate treatment dose is routinely made by those ofordinary skill in the art without undue experimentation, especially inlight of the dosage information and assays disclosed herein, as well asthe pharmacokinetic data observed in animals or human clinical trials.

[0168] Appropriate dosages may be ascertained through the use ofestablished assays for determining blood clotting levels in conjunctionwith relevant dose-response data. The final dosage regimen will bedetermined by the attending physician, considering factors that modifythe action of drugs, e.g., the drug's specific activity, severity of thedamage and the responsiveness of the patient, the age, condition, bodyweight, sex and diet of the patient, the severity of any infection, timeof administration, and other clinical factors. As studies are conducted,further information will emerge regarding appropriate dosage levels andduration of treatment for specific diseases and conditions.

[0169] In gene therapy embodiments employing viral delivery, the unitdose may be calculated in terms of the dose of viral particles beingadministered. Viral doses include a particular number of virus particlesor plaque forming units (pfu). For embodiments involving adenovirus,particular unit doses include 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², 10¹³ or 10¹⁴ pfu. Particle doses may be somewhat higher (10to 100-fold) due to the presence of infection defective particles.

[0170] It will be appreciated that the pharmaceutical compositions andtreatment methods of the invention may be useful in fields of humanmedicine and veterinary medicine. Thus, the subject to be treated may bea mammal, preferably human or other animal. For veterinary purposes,subjects include, for example, farm animals including cows, sheep, pigs,horses and goats, companion animals, such as dogs and cats, exoticand/or zoo animals, laboratory animals including mice rats, rabbits,guinea pigs and hamsters, and poultry such as chickens, turkeys, ducks,and geese.

I. EXAMPLES

[0171] The present invention is described in more detail with referenceto the following non-limiting examples, which represent preferredembodiments of the invention. Those of skill in the art will understandthat the techniques described in these examples represent techniquesdescribed by the inventors to function well in the practice of theinvention, and as such constitute preferred modes for the practicethereof. However, it should be appreciated that those of skill in theart should in light of the present disclosure, appreciate that manychanges can be made in the specific methods that are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Transient Expression of Human Factor IX

[0172] Construction and Transient Expression of Factor IX Constructs.The recombinant human factor IX cDNA in the expression vector pCMV5 wasgenerously provided by Darrel Stafford (Univ. North Carolina). The EcoRIfragment of the cDNA insert was excised from the pCMW5-human factor DCexpression vector and subcloned into pBluescript SK II for mutagenesis.Mutations are constructed by PCR using a high fidelity DNA polymerase(pfu) followed by Dpn I digestion of the parental plasmid (QuikChangeMutagenesis Kit, Strategene). Following transformation, clonescontaining the desired mutation(s) are selected by DNA sequencing. TheEcoRI fragment is excised from plasmids with the desired mutation(s) andsubcloned back into the pCMV5 expression vector. Proper orientation ofthe constructs for expression is confirmed by restriction digest withBam HI/Bgl II. Mutant factor IX cDNA constructs are screened for proteinexpression and initial characterization as described below.

[0173] Initial Characterization of Transiently Expressed Factor IXProteins. The use of initial characterization of constructs in transienttransfections will allow one of skill in the art to monitor and modifythe results of the mutagenesis strategy to assist in the selection ofstable cell lines and purification of the mutant protein.

[0174] The pCMV5-HuFIX wild-type construct was transiently transfectedinto 293 cells with Lipofectin (Gibco-BRL). Following transfection,cells were incubated in serum free media (SFM) containing 10 μg/mLVitamin K for 48 hr. The SFM was harvested, concentrated 10-12 fold byCentricon-30, and assayed for clotting and intrinsic tenase activity.

[0175] Clotting activity and intrinsic tenase activity were easilydetected in the media, with no significant background detected withmock-transfected cells. Factor IX antigen concentration was determinedusing a “sandwich” type ELISA with affinity purified sheep anti-humanfactor IX polyclonal antibody (Hematologic Technologies) as the captureantibody, and a horseradish peroxidase-conjugated affinity purifiedsheep anti-human factor IX antibody (Enzyme Research) to detect theimmobilized antigen. The assay demonstrated a linear relationship(log-log plots) from 0.1 to 100 μg/mL human factor IX, and estimatedfactor IX concentrations in the 0.6 μg/mL range (10-12 nM) followingtransient transfection of pCMV5-factor IX.

[0176] Factor X activation by the mutant proteins may be determined inthe intrinsic tenase assay following activation from the zymogen formwith factor XIa. In this method, thrombin-activated factor VIIIa (finalconcentration 0.5 nM) is added to a reaction containing 5 nM factor IXa,5% (v/v) rabbit brain cephalin, 300 nM factor X, and increasingconcentrations of heparin in 0.15 M NaCl, 20 mM HEPES, pH 7.4, 2 mMCaCl₂, and 0.1% PEG-8000. The reaction is sampled (50 μl) at 15, 30, 45,and 60 sec into 10 μl of 0.25 M EDTA, pH 8.0. The chromogenic substrate,S-2765, is then added at 300 μM and the amount of factor Xa generateddetermined by comparison of the rate of cleavage with a standard curve[Sheehan and Lan, Blood, 92(5): 1617-1625 (1998)]. Alternatively, thischromogenic assay for factor Xa generation can be made more quantitativefor factor IXa (0.5 nM) by performing it in the presence of excessfactor VIIIa (5 nM).

[0177] Coagulant activity of the mutant proteins is determined by anAPTT in factor IX deficient plasma, with comparison to a standard curve[Bajaj et al., Meth Enzymol, 222: 96-128 (1993)].

Example 2 Stable Expression and Purification of Human Factor IX

[0178] The present example provides methods for the recombinantexpression and purification of recombinant human factor IX. Stable celllines expressing recombinant factor IX are selected for large scaleproduction of protein, and factor IX is purified to homogeneity fromserum-free conditioned media.

[0179] Stable cell lines expressing recombinant human factor IX wereobtained by co-transfecting ScaI-linearized pCMV5-huFIX and pSV2neoplasmids into 293 cells by the calcium phosphate method, and selectingclones by limiting dilution in G418. Cell lines expressing high levelsof the recombinant factor IX were determined by ELISA and/or Westernblot. Cell lines with the highest expression levels were then expandedfor large-scale culture in T-225 cm² flasks. Upon reaching confluence,the growth media (50% DME/50% F-12/10% FCS) was removed, the monolayerswashed extensively with SFM, and replaced with SFM supplemented withinsulin-transferrin-sodium selenite (Sigma) and 10 μg/mL vitamin K.Conditioned media was collected every 48 hours for 10 days. Benzamidinewas added to a final concentration of 5 mM, and the conditioned mediafrozen at −20° C. to −25° C. Upon thawing, the conditioned media waspooled, filtered, and subjected to barium chloride precipitation [Coteet al., J Biol Chem, 269(15):11374-80 (1994)]. The precipitate wasdissolved in 0.2 M EDTA, and the eluate dialyzed overnight beforeapplication to a Mono Q HR 5/5 column (0.15 M NaCl, 20 mM HEPES, pH 7.4,0.1% PEG-8000). Human factor IX was eluted with a calcium chloridegradient (0-45 mM) and concentrated in a Centricon-30.

[0180] Purity of factor IX proteins was assessed by SDS-PAGE with silverstaining, and the factor IX concentration was determined by A280(1%=13.3) and ELISA. The human factor IX isolated above demonstratedhigh purity by 10% SDS-PAGE with silver staining (FIG. 1), high specificclotting activity (187 U/mg), and an overall yield of approximately 30%by ELISA. Additionally, elimination of factor IX antigen with lowclotting activity (partial degradation or incomplete carboxylation) wasdetected by Western blot following 2 M NaCl elution of the Mono Q columnfollowing the calcium chloride gradient.

[0181] Other studies maybe used to determine the activity of therecombinant factor IXa. Recombinant factor IX may be activated byincubation with human factor XIa (Enzyme Research) at a molar ratio of100:1 in 150 mM NaCl, 20 mM HEPES, 2 mM CaCl2, pH 7.4. for 2 hr at 37°C. Activation is monitored by SDS-PAGE, and factor IXa specific activityestimated by active site titration with antithrombin [Chang et al., JBiol Chem, 273(20): 12089-94 (1998)].

[0182] Using the methods described in the present example, it ispossible to purify highly active recombinant factor IX to homogeneityfor detailed analysis of enzymatic and binding properties.

Example 3 Three-Dimensional Model of Factor IX

[0183] A three-dimensional structure of human factor IXa was obtained byhomology modeling with SWISS-MODEL, using the crystal structures ofrecombinant human factor IXa complexed with p-aminobenzamidine (1RFN),porcine factor IXa complexed with D-FPR-chloromethylketone (1PFX), humanfactor VIIa with soluble tissue factor (1DAN), and human factor Xacomplexed with the synthetic inhibitor FX-2212A (1XKA, 1XKB) astemplates [Hopfner et al., Structure Fold Des, 7(8): 989-96 (1999);Brandstetter et al., Proc Nat'l Acad Sci USA, 92(21): 9796-800 (1995);Banner et al., Nature, 380(6569): 41-6 (1996)]. The availability of athree-dimensional model of the protease is extremely helpful forplanning and modification of the mutagenesis strategy.

Example 4 Expression of Mutant Human Factor IX

[0184] The factor IX R233A construct was transiently expressed in 293cells, concentrated by Centricon-30, and tested for clotting andintrinsic tenase activity. Initial experiments demonstrated roughlyequivalent clotting activity to wild-type factor IX in factorIX-deficient plasma. After activation with factor XIa in conditionedmedia, the inhibitory effects of heparin on intrinsic tenase activitywere tested in the presence of excess factor VIIIa. The relationshipbetween clotting activity and intrinsic tenase activity in the absenceof inhibitors was roughly proportionate for both recombinant proteases.Compared to wild-type factor IXa, the mutant R233A demonstrated markedlyreduced inhibition by heparin.

[0185] Although a KJ cannot be calculated from the transienttransfection data, a marked increase in the residual activity in theplateau phase was noted for the mutant R233A (˜65%) relative towild-type factor IXa (˜15%) (FIG. 2). Similar effects on heparininhibition were noted in transient transfection experiments with thefactor IX K241A construct.

Example 5 Comparison of In Vitro Antithrombin-Independent Inhibition ofWild-Type and Mutant R-233A Human Factor IXa by Unfractionated Heparin

[0186] The present example demonstrates the resistance of the purifiedfactor IXa mutant R233A to antithrombin independent inhibition byunfractionated heparin. Factor Xa generation by 5 nM wild-type () orR233A (◯) factor IXa in the intrinsic tenase complex (0.5 nM factorVIIIa. 5% rabbit brain cephalin, 300 nM factor X and 2 mM CaCl₂) wasdetermined in the presence of increasing amounts of unfractionatedheparin (as described in Example 1; also, see FIG. 4). The data were fitby nonlinear regression to the equation for partial, uncompetitiveinhibition. The mutant factor IXa R233A demonstrates increasedresistance to inhibition by heparin, as demonstrated by the significantreduction in maximal inhibition (increase in enzymatic activity)observed relative to wild-type factor IXa.

Example 6 Kinetic Analysis of Activation by Human Factor XIa onWild-Type and Recombinant Human Factor IXa Mutant Proteins

[0187] 293 cells were co-transfected with pSV2neo and pCMV5-huFIXconstructs, and stable cell-lines expressing the recombinant humanfactor IX proteins were selected by resistance to the antibiotic G418.Human factor A. H92A, R233A, and K241A were purified to homogeneity fromconditioned media. Clotting activity was determined in an APTT assayperformed in factor IX deficient plasma (Table D). Wild-type and factorIX R233A were activated to factor IXa with human factor XIa. Analysis ofthe time course for factor IX activation by human factor XIa on an 10%SDS-PAGE gel demonstrated no significant difference between wild-typefactor IX and the mutant R233A.

[0188] Following activation to factor IXa, the ability of heparin toinhibit factor X activation by the recombinant proteins was examined forboth the factor IXa-phospholipid (in the presence of 30% ethyleneglycol) and intrinsic tenase complex (factor VIIIa-factorIXa-phospholipid) (FIGS. 4A and 4B). Factor IXa R233A demonstratedresistance to inhibition by heparin relative to wild-type factor IXaunder both assay conditions, suggesting that this mutation adverselyaffected the interaction of factor IXa with heparin. TABLE D RelativeHeparin Affinity and Clotting Activity of Recombinant Factor IX MutantProteins. Heparin-Sepharose Clotting Activity Protein Elution (M NaCl)(% normal) Plasma-derived factor 0.49 N.D. IX Plasma-derived factor 0.58100% IXa Wild-type factor IXa 0.58  90% Factor IXa H92A 0.54  93% FactorIXa R170A 0.56 N.D. Factor IXa R233A 0.33  36% Factor IXa K241A 0.56 55% # activated with 25 nM human factor XIa (200:1, substrate:enzyme)for 2 hr at 37° C. and then applied to a heparin-sepharose column # (1mL) at a flow rate of 0.5 mL/min. The column was washed with 10 mL of0.05 M NaCl, 20 mM HEPES, pH 7.4, 0.1% PEG-8000, and 5 mM EDTA, #followed by elution with a 0.05 to 1.0 M NaCl gradient at 1 mL/min.Clotting activity was determined in an APTT assay performed by #addition of the zymogen from (factor IX) to factor IX deficient plasma(with comparison to a standard curve).

Example 7 Heparin Binding Affinity of Wild-Type and Recombinant HumanFactor IXa Mutant Proteins

[0189] The relative affinity of the recombinant proteins for heparin wasassessed by the position of elution from a heparin-sepharose column inresponse to a NaCl gradient. Heparin-protein interactions are generallydominated by electrostatic forces, thus, this assay is a reasonablesurrogate for direct binding assays. The interaction of the homologouscoagulation protease thrombin with heparin has been examined in detail,demonstrating the predominant contribution of electrostatic forces[Olson et al, J Biol Chem, 266(10): 6342-52 (1991)]. Likewise, theeffect of mutations on the affinity of recombinant thrombins for heparinhas previously been assessed by NaCl elution from heparin-sepharose, andcorrelated with the functional effects of these mutations on inhibitionby antithrombin-heparin [Sheehan et al., Proc Nat'l Acad Sci USA;91(12): 5518-22 (1994)].

[0190] Substitution of alanine for homologous residues in human factorIXa resulted in an elution of the recombinant factor IXa from theheparin sepharose at a lower concentration of NaCl, consistent with areduction in heparin affinity.

[0191] Plasma-derived factor IX and IXa eluted from heparin-sepharose at0.49 and 0.58 M NaCl, respectively, suggesting that the activatedprotease binds with higher affinity than the zymogen form. Wild-typefactor IXa eluted similarly to plasma-derived factor IXa, suggestingthat any differences in post-translational modifications between theseproteins did not affect heparin binding. The remainder of therecombinant factor IXa proteins demonstrated either modest or markedreduction in apparent heparin affinity (elution at lower NaClconcentration). The first group included factor IXa H92A, R170A, andK241A, which demonstrated modest reductions in apparent heparinaffinity. The second group included factor IXa IR233A, whichdemonstrated a marked reduction in apparent heparin affinity (Table D).These data demonstrate that the selected mutations (especially R233A)reduce apparent heparin affinity, suggesting that these residuescontribute to a heparin-binding exosite on factor IXa.

[0192] The modest effect of single alanine substitutions is notunexpected given the electrostatic, multivalent nature ofheparin-protease binding [Olson et al., supra (1991)], and either chargereversal (substitution of glutamate/aspartate) or combinatorial mutantsare expected to significantly enhance this effect. The relative effectsof these mutations on apparent heparin affinity of factor IXa suggestthat the heparin-binding exosite maps to the carboxyl-terminus region ofthe protease.

Example 8 Clotting Activity of Wild-Type and Recombinant Human FactorIXa Mutant Proteins

[0193] Clotting activity was examined in a modified activated partialthromboplastin time for both plasma-derived and recombinant factor IXa.Consistent with previous reports, wild-type factor IXa demonstratedapproximately 90% of plasma-derived factor IXa clotting activity. Thisresult may be secondary to the presence of a minor factor IX form (4-5%)in which the prosequence has not been cleaved [Bajaj et al., J BiolChem, 272(37): 23418-26 (1997)]. Thus, the wild-type factor IXa clottingactivity represents the appropriate control for comparison of therecombinant factor IXa mutant proteins. Factor IXa R233A and K241Ademonstrated moderate reductions in clotting activity relative towild-type, while factor IXa H92A had similar clotting activity towild-type factor IXa. Likewise, factor IXa R170A was reported to haveincreased clotting activity relative to wild-type or plasma-derivedfactor IXa [Chang et al., J Biol Chem, 273(20): 12089-94 (1998)]. Thus,the effect of amino acid substitutions on relative heparin affinity canclearly be dissociated from effects on clotting activity.

[0194] While the methods and compositions herein have been described interms of preferred embodiments, it will be apparent that variations maybe applied to the methods and/or compositions without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that assays that are physiologically related may besubstituted for the assays described herein while still producing thesame or similar results. All such similar substitutes and modificationsapparent to those of skill in the art are deemed to be within the scopeof the invention as defined by the appended claims.

[0195] The present specification cites to certain scientific journalreferences and patents that, to the extent that they provide exemplaryprocedural or other information supplemental to that set forth herein,are specifically incorporated herein by reference.

1 2 1 1389 DNA Homo sapiens 1 atgcagcgcg tgaacatgat catggcagaatcaccaagcc tcatcaccat ctgcctttta 60 ggatatctac tcagtgctga atgtacagtttttcttgatc atgaaaacgc caacaaaatt 120 ctgaatcggc caaagaggta taattcaggtaaattggaag agtttgttca agggaacctt 180 gagagagaat gtatggaaga aaagtgtagttttgaagaac cacgagaagt ttttgaaaac 240 actgaaaaga caactgaatt ttggaagcagtatgttgatg gagatcagtg tgagtccaat 300 ccatgtttaa atggcggcag ttgcaaggatgacattaatt cctatgaatg ttggtgtccc 360 tttggatttg aaggaaagaa ctgtgaattagatgtaacat gtaacattaa gaatggcaga 420 tgcgagcagt tttgtaaaaa tagtgctgataacaaggtgg tttgctcctg tactgaggga 480 tatcgacttg cagaaaacca gaagtcctgtgaaccagcag tgccatttcc atgtggaaga 540 gtttctgttt cacaaacttc taagctcacccgtgctgagg ctgtttttcc tgatgtggac 600 tatgtaaatc ctactgaagc tgaaaccattttggataaca tcactcaagg cacccaatca 660 tttaatgact tcactcgggt tgttggtggagaagatgcca aaccaggtca attcccttgg 720 caggttgttt tgaatggtaa agttgatgcattctgtggag gctctatcgt taatgaaaaa 780 tggattgtaa ctgctgccca ctgtgttgaaactggtgtta aaattacagt tgtcgcaggt 840 gaacataata ttgaggagac agaacatacagagcaaaagc gaaatgtgat tcgagcaatt 900 attcctcacc acaactacaa tgcagctattaataagtaca accatgacat tgcccttctg 960 gaactggacg aacccttagt gctaaacagctacgttacac ctatttgcat tgctgacaag 1020 gaatacacga acatcttcct caaatttggatctggctatg taagtggctg ggcaagagtc 1080 ttccacaaag ggagatcagc tttagttcttcagtacctta gagttccact tgttgaccga 1140 gccacatgtc ttcgatctac aaagttcaccatctataaca acatgttctg tgctggcttc 1200 catgaaggag gtagagattc atgtcaaggagatagtgggg gaccccatgt tactgaagtg 1260 gaagggacca gtttcttaac tggaattattagctggggtg aagagtgtgc aatgaaaggc 1320 aaatatggaa tatataccaa ggtatcccggtatgtcaact ggattaagga aaaaacaaag 1380 ctcacttaa 1389 2 462 PRT Homosapiens 2 Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Ser Leu IleThr 1 5 10 15 Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr ValPhe Leu 20 25 30 Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys ArgTyr Asn 35 40 45 Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu ArgGlu Cys 50 55 60 Met Glu Glu Lys Cys Ser Phe Glu Glu Pro Arg Glu Val PheGlu Asn 65 70 75 80 Thr Glu Lys Ile Thr Glu Phe Trp Lys Gln Tyr Val AspGly Asp Gln 85 90 95 Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys LysAsp Asp Ile 100 105 110 Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe GluGly Lys Asn Cys 115 120 125 Glu Leu Asp Val Thr Cys Asn Ile Lys Asn GlyArg Cys Glu Gln Phe 130 135 140 Cys Lys Asn Ser Ala Asp Asn Lys Val ValCys Ser Cys Thr Glu Gly 145 150 155 160 Tyr Arg Leu Ala Glu Asn Gln LysSer Cys Glu Pro Ala Val Pro Phe 165 170 175 Pro Cys Gly Arg Val Ser ValSer Gln Thr Ser Lys Leu Thr Arg Ala 180 185 190 Glu Ala Val Phe Pro AspVal Asp Tyr Val Asn Pro Thr Glu Ala Glu 195 200 205 Thr Ile Leu Asp AsnIle Thr Gln Gly Thr Gln Ser Phe Asn Asp Phe 210 215 220 Thr Arg Val ValGly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp 225 230 235 240 Gln ValVal Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile 245 250 255 ValAsn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly 260 265 270Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu 275 280285 His Thr Glu Gln Lys Arg Asn Val Ile Arg Ala Ile Ile Pro His His 290295 300 Asn Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu305 310 315 320 Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr ProIle Cys 325 330 335 Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys PheGly Ser Gly 340 345 350 Tyr Val Ser Gly Trp Ala Arg Val Phe His Lys GlyArg Ser Ala Leu 355 360 365 Val Leu Gln Tyr Leu Arg Val Pro Leu Val AspArg Ala Thr Cys Leu 370 375 380 Arg Ser Thr Lys Phe Thr Ile Tyr Asn AsnMet Phe Cys Ala Gly Phe 385 390 395 400 His Glu Gly Gly Arg Asp Ser CysGln Gly Asp Ser Gly Gly Pro His 405 410 415 Val Thr Glu Val Glu Gly ThrSer Phe Leu Thr Gly Ile Ile Ser Trp 420 425 430 Gly Glu Glu Cys Ala MetLys Gly Lys Tyr Gly Ile Tyr Thr Lys Val 435 440 445 Ser Arg Tyr Val AsnTrp Ile Lys Glu Lys Thr Lys Leu Thr 450 455 460

What is claimed is:
 1. A mutant human factor IX comprising a mutation inthe heparin binding domain which decreases its affinity for heparin ascompared to wild-type human factor IX.
 2. The mutant human factor IX ofclaim 1, wherein said mutation is a mutation of the amino acid residue233, 230, 239, 241, 87, 91, 98, 101, or 92 of wild-type human factor IX.3. The mutant human factor IX of claim 2, wherein said mutation furthercomprises a substitution of arginine 170 of the wild-type human factorIX for an alanine.
 4. A mutant human factor IX having a mutation of theamino acid located at residue number 233 of wild-type human factor IX,wherein said mutation decreases the affinity of said mutant human factorIX for heparin as compared to wild-type human factor IX.
 5. The mutanthuman factor IX of claim 4, wherein said mutation is a substitution ofthe arginine at position 233 to any other amino acid.
 6. The mutanthuman factor IX of claim 5, wherein arginine at position 233 issubstituted with an alanine.
 7. A method of treating a subject havinghemophilia comprising administering to said subject a compositioncomprising a mutant human factor IX of claim 1, in an amount effectiveto promote blood clotting in said subject.
 8. The method of claim 7,wherein said mutant human factor IX comprises a mutation of the aminoacid located at residue number 233 of wild-type human factor IX.
 9. Themethod of claim 8, wherein the mutant human factor IX comprises amutation of arginine 233 to an alanine
 233. 10. The method of claim 7,further comprising administering to said subject a compositioncomprising one or more additional blood clotting factors other than saidmutant human factor IX.
 11. The method of claim 7, wherein saidhemophilia is hemophilia B.
 12. A method of treating hemophilia in amammal comprising: (a) providing an expression construct comprising apolynucleotide encoding a mutant factor IX according to any one ofclaims 1 through 6, operably linked to a promoter; and (b) administeringan amount of said expression construct to a mammal wherein said mutantfactor IX is expressed at levels having a therapeutic effect on saidmammal.
 13. The method of claim 12, wherein said therapeutic effect isan increased resistance of factor IX to inhibition by heparin.
 14. Themethod of claim 12, wherein said therapeutic effect is a decrease in theblood clotting time of said mammal as compared to the blood clottingtime of said mammal in the absence of said expression construct.
 15. Themethod of claim 12, wherein said expression construct comprises a viralvector selected from the group consisting of an adenovirus, anadeno-associated virus, a retrovirus a herpes virus, a lentivirus and acytomegalovirus.
 16. The method of claim 12, wherein said expressionconstruct is administered by injecting said vector into at least twosites in the mammal.
 17. The method of claim 12, wherein said expressioncontrol element is selected from the group consisting of acytomegalovirus immediate early promoter/enhancer, a skeletal muscleactin promoter, and a muscle creatine kinase promoter/enhancer.
 18. Arecombinant host cell stably transformed or transfected with apolynucleotide encoding a mutant human factor IX of any one of claims 1through 6 in a manner allowing the expression in said host cell of saidmutant human factor IX.
 19. A pharmaceutical composition comprising amutant human factor of any one of claims 1 through 6 and apharmaceutically acceptable carrier, excipient, or diluent.
 20. Apharmaceutical composition comprising: i) an expression constructcomprising a vector having an isolated polynucleotide encoding a mutanthuman factor IX of any one of claims 1 through 6 and a promoter operablylinked to said polynucleotide; and ii) a pharmaceutically acceptablecarrier, excipient, or diluent.